Linker molecule and use thereof in methods for purifying peptides

ABSTRACT

The present invention relates to a method for the purification of peptides which are produced by solid phase peptide synthesis (SPPS) and corresponding linker molecules for use in said method. Optionally, the peptide may be modified while bound via said linker molecule on a purification support.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a Continuation-in-Part of U.S. patent application Ser. No.16/073,794, filed Dec. 26, 2018, which is the US National Stage ofInternational Patent Application No. PCT/EP2017/051932, filed on Jan.30, 2017, and which claims priority to German Patent Application No. 102016 101 606.3, filed Jan. 29, 2016. The foregoing patent applicationsare incorporated by reference herein in their entirety.

FIELD OF THE INVENTION

The present invention relates to a method for the purification ofpeptides produced by solid phase peptide synthesis (SPPS) andcorresponding linker molecules for use in this method.

BACKGROUND

Solid phase peptide synthesis is a well-known method for the productionof peptides. Apart from the synthesis of the peptides, theirpurification is also an essential process step.

A widely used method for the purification of peptides is the preparativehigh performance liquid chromatography (HPLC). Disadvantageous at thismethod is the poor scalability with regard to the desired productionquantities, so that different quantities cannot be produced with one andthe same system; this causes relatively high acquisition costs for thecorresponding complex devices. A further disadvantage is that thecorrect analytical assessment of the individual fractions requiresrelatively extensive knowledge; additionally, there is the consumptionof solvents and column material (solid phase) during operation.

Therefore, methods that are cheaper and less prone to faults would beadvantageous for reducing the costs of peptide production.

EP 0 552 368 A1 describes a method for the purification of peptides inwhich a so-called linker is on the one hand covalently bound to theN-terminal end of the synthesized full-length peptide and on the otherhand covalently bound by reaction to thioether of a thiol group withfunctionalized diatomaceous earth. The full-length peptide is thusimmobilized and can be purified. The full-length peptide is thenreleased under basic conditions. However, the method is not suitable forthiol-containing peptides such as those comprising the amino acidcysteine or penicillamine. Furthermore, there is the disadvantage thatthe solid phase used for purification (in this case diatomaceous earth)is not intended or suitable for reuse.

EP 2 501 711 B1 proposes an analogous method in which the linker isbound to a solid phase (synthetic hydrophilic polymer, e.g. PEGA) viathe N-terminal end of the synthesized full-length peptide and via a1,3-dipolar cycloaddition between an azide (—N₃) and an alkyne (Huisgenreaction). A disadvantage of this method is here the necessity of addingcopper or copper-containing compounds to perform the 1,3-dipolarcycloaddition. Many peptides complex copper, particularly thosecomprising sulphur, i.e. comprising methionine and/or cysteine; arginineand lysine can also bind to copper. The copper is therefore difficult toremove and due to the toxicity of the remaining copper the method is notapplicable in all cases, especially not for the purification of peptidetherapeutics. Another disadvantage is that the solid phase used forpurification is not intended or suitable for reuse.

Linear unmodified peptides show low in vivo stability due to the fastdegradation by proteases and peptidases. Additionally, they show nocell-permeability and fast clearance rates towards the kidney in mammalbodies (Witt et al. (2001), Peptides (22), 2329-43). Mainly twomodifications are used to improve in vivo peptide stability and potency:Lipidation with fatty acids and cyclisation of linear peptides to formmacrocycles.

Cyclic peptides have many advantages over linear peptides. They aresignificantly more resistant to both exo- and endoproteases. Also, theyshow superior binding affinities to desired protein targets most likelybecause they display a more rigid and arch-like binding motive towardstarget receptors. Successful cyclic peptide drugs are the anti-cancerdrug octreotide (Sandostatin®), the immunosuppressant ciclosporin(Sandimmun®, Sandimmun®, Neoral®) or the neuropeptide hormone oxytocine(Syntocinon® or others). Methods to form peptidic cycles can beseparated in two main categories: a) one-component-system, in whichpeptides can be cyclized by utilizing their intrinsic functional groupsto form cycles connected by e.g. amides or disulfides. By incorporationof unnatural amino acids other covalent connections can be created. b)Two-component-system, in which the functional groups of the peptides areused in reactions with a bridging scaffold. Organic molecules can beused, that form together with the peptides mono-, bi- or multi-cycles.

When SPPS is used peptides are either cyclized on the synthetic resin orafter detachment of peptide from the synthetic resin in solution. Bothmethods have their pitfalls. Modification on solid support demandsorthogonally protected amino acids, that are expensive and need extrasteps to be deprotected before modification. As an advantage, the solidsupport helps to prevent undesired oligo- or polymerization peptides dueto pseudo ultra-high dilution. Furthermore, excess of reagent can beused, since it easily can be washed away. Cyclisation in solution hasthe downside of being notorious to side reactions such as oligo- orpolymerization and excess of oxidation reagents are tedious to beremoved. Consequently, disulfide cyclizations are performed in highdiluted solution what limits production scale-up.

SUMMARY

It is therefore the object of the invention to provide a method for thepurification of peptides that does not require a complete HPLC systemand is also suitable for sulphur-containing or copper-binding peptides.In addition, it is the object of the invention to provide a methodsuitable for the purification of peptides that allows a regeneration andreuse of the solid phase used for purification. Furthermore, it is theobject of the invention to provide a compound that allows bindingbetween the N-terminal amino group of a full-length peptide and a solidphase.

The present invention utilizes linker molecules that enable theimmobilization of peptide precursors followed by subsequent modificationof peptides while they are immobilized on the purification/modificationsolid support. In contrast to other methods especially to those that usemodification in solution the present invention removes perturbatingpeptidic truncations and excess of organic molecules for modificationsimultaneously and is thus a method for modification and at the sametime purification of desired peptides.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a schematic representation of the method according to theinvention to illustrate the same. i) SPPS with acetylation after eachcoupling, n-fold repetition; ii) addition of molecule X1-L-X2, iii)immobilization; iv) washing; v) basic cleavage; vi) filtration; vii)regeneration. 1: purification resin, 2: capture molecule, 3: nativepeptide (desired product), 4: truncated sequences, x=1 to n, n:full-length peptide, n-x: truncated sequences.

FIG. 2 shows the absorption at 278 nm when measuring the supernatantafter cleaving off the peptide (compound 3 in FIG. 6) with 0.5 vol. %N₂H₄ to demonstrate the reversibility of the hydrazone bond fromexample 1. Abs.: Absorption of the supernatant of peptide-loadedaldehyde-modified agarose support at 278 nm. t: Time elapsed after theaddition of a 0.5% hydrazine solution in minutes (min). n: Amount ofpeptide in the supernatant in nmol calculated on the basis ofabsorption. Dashed line: Non-linear regression according to Hill withthe formula: y=0.142*x/(3.16+x), R²=92, t_(1/2)=3.16 min.

FIG. 3 shows chromatograms of the individual phases upon purificationaccording to the invention of a peptide after Native Chemical Ligation(NCL) from Example 2. Chromatogram 1 shows the desired product (P) inthe reaction mixture of the NCL after 24 hours. Chromatogram 2 shows thesupernatant after 30 minutes of immobilization. Chromatogram 3 shows thedesired product (P) after 30 minutes of washing and cleavage. P: Peak ofthe desired product.

FIG. 4 shows chromatograms upon purification according to the inventionof peptides 7-13 after solid phase peptide synthesis from example 4. Thechromatograms, which are labeled with supernatant, show the impuritieswhich could be separated by the method. FM=capture molecule according toinvention; (a) peptide 7 (Tau1); (b) peptide 8 (Tau2); (c) peptide 9(GNRH), (d) peptide 10 (Magainin); (e) peptide 11 (Terts72Y); (f)peptide 12 (bivalrudine); (g) peptide 13 (TAT), (h) peptide 14 (researchpeptide). Each upper diagram shows the peptide after synthesis and eachlower diagram shows the peptide after purification.

FIG. 5 shows a schematic representation of the method according to theinvention to illustrate the same. FIG. 5 shows an alternativerepresentation to FIG. 1.

SEQ ID Nos: 1 to 10 show peptides from examples 1, 2 and 4.

FIG. 6 shows Scheme 3: Reversibility of the hydrazone bond between apeptide and a purification resin. i) 0.1 M NH₄OAc, pH=4, 0.1 M PhNH₂, 30min; ii) 0.5% N₂H₄, 5 mM TCEP; the peptide sequence (white characters inblack area) is assigned to SEQ ID NO: 1.

FIG. 7 shows Scheme 4: Purification of a peptide after native chemicalligation i) 0.1 M Na₂HPO₄, 3 M Gdn*HCl, 20 mM TCEP, 50 mM MesNa, 1%(v/v) PhSH, pH=7, 15 h; ii) Addition sepharose resin and 2-fold vol. of0.1 M NH₄OAc pH=2.5, pH=4, 30 min; iii) Washing H₂O, EtOH; iv) N₂H₄, 4mM TCEP, 30 min. The peptide sequences are assigned to SEQ ID NO: 1(white characters in black area) and SEQ ID NO: 2 (“YENDRIK” in whitearea).

FIG. 8 shows Scheme 5: Purification according to the invention of apeptide mixture after solid phase peptide synthesis (SPPS) i) SPPS withacetylation after each coupling, n-fold repetition; ii) Addition ofmolecule 2; iii) Cleavage with TFA; iv) Immobilization on purificationresin by addition of Ph-NH₂ at pH 3-4; v) Washing with water and buffer;vi) Base 5% NH₄OH; vii) Regeneration of the purification resin 1 byaddition of H₂O/acetone/TFA (49,5/49,5/1). 1: purification resin, 2:capture molecule, 3: native peptide (desired product), 4: truncatedsequences, x=1 to n, n: full-length peptide, n-x: truncated sequences.

FIG. 9 shows Scheme 6: Functionalization of agarose beads i) NaOH,NaBH₄, H₂O, r.t, o/n, 18 h; ii) 20 mM NaIO₄, H₂O, r.t, 1 h.

FIG. 10 shows Scheme 7: Synthesis of the base-labile linker i) N₂H₄*H₂O;ii) 2.2 eq. pNO₂PhCO₂Cl; iii) mCPBA; n: peptide.

FIG. 11 shows an example of the inventive peptidemodification/purification of peptide P1 (H-CRVPGDAHHADSLC-NH₂) by usageof inventive linker molecule (14). A=absorption (210 nm), B=time/min;1.) Chromatogram of trail cleavage of crude peptide sample before linker(14) coupling to P1. 2.) Chromatogram of P1 after cyclization viadisulfide formation and purification.

FIG. 12 shows an example of the inventive peptidemodification/purification of peptide P2 (H-VRCPGAAHHADSLC-NH₂) by usageof inventive linker molecule 14. A=absorption (210 nm), B=time/min; 1.)Chromatogram of trail cleavage of crude peptide sample before linker(14) coupling to P2. 2.) Chromatogram of P2 after cyclization viadisulfide formation and purification.

FIG. 13 shows an example of the inventive peptidemodification/purification of peptide P3 (H-VRVPGCAHCADSLY-NH₂) by usageof inventive linker molecule 14. A=absorption (210 nm), B=time/min; 1.)Chromatogram of trail cleavage of crude peptide sample before linker(14) coupling to P3. 2.) Chromatogram of P3 after cyclization viadisulfide formation and purification.

FIG. 14 shows an example of the inventive peptide purification ofpeptide P4 HIV reverse transcriptase (159-173) H-ELRQHLLRWGLTTPD-NH₂ byusage of inventive linker molecule 14. A=absorption (210 nm),B=time/min; 1.) Chromatogram of trail cleavage of crude peptide samplebefore linker (14) coupling to P4. 2.) Chromatogram of P4 afterpurification.

BRIEF DESCRIPTION OF THE DESCRIBED SEQUENCES

The amino acid sequences provided herewith are shown using standardthree letter code for amino acids, as defined in 37 C.F.R. 1.822. TheSequence Listing is submitted as an ASCII text file named95083_355_1001_seglist, created Mar. 21, 2021, about 4 KB, which isincorporated by reference herein.

DETAILED DESCRIPTION

In a first aspect of the present invention, this object is achieved by acompound of the general formula

X₁-L-X₂  (1), wherein

X₁ is selected from

-   -   wherein each R¹ and R² is independently from each other selected        from H or B, wherein at least R¹ or R² is B, wherein    -   R³ is selected from H or B,        -   wherein B is an acid labile amine protecting group, wherein    -   R⁴ is selected from H, C₁-C₁₂-alkyl or aryl, wherein the        aldehyde or keto group may be protected by an acid labile        protecting group,

L is selected from functional linkers, that are cleavablenucleophilically from X₂ under basic conditions, in particular L is ofthe form -T-U—, wherein

-   -   T is a spacer between X₁ and U, wherein in particular T is        selected from substituted or unsubstituted —C₁-C₁₂-alkyl-, in        particular C₁-C₆-alkyl, in particular C₁-C₃-alkyl,        —R⁵—C(═O)—NH—R⁶—, —R⁵—C(═O)—O—R⁶—, —R⁵—C(═O)—O—, —C(═O)—O—R⁶—,        —C(═O)—NH—R⁶—, —C(═O), —C(═O)—O—, —R⁵-phenyl-R⁶—, —R⁵-phenyl-,        -phenyl-R⁶—, -phenyl-,        -   wherein R⁵ and R⁶ are independently from each other selected            substituted or unsubstituted C₁-C₁₂-alkyls, in particular            C₁-C₆ alkyls, particularly C₁-C₃ alkyls, and wherein    -   U is the cleavage activating part of the functional linker,        wherein the activating part is formed to stabilize an anion        formed during an basic cleavage from X₂,

X₂ is of the form —Y—Z, wherein

-   -   Y is selected from —O—C(═O)— or —S(═O)₂—, and    -   Z is an electron-withdrawing leaving group.

In the context of the present invention, the term “cleavage activatingpart” of a molecule relates to a structural element of a reactivefunction.

“Reactive function” relates to a compound that can be excited(activated) to generate a reactive species. This can be a catalyst or achange in pH value, for example. The reactive species is able to form acovalent bond, for example a carbamate bond, in a short time with asuitable reaction partner. The reactive function thus comprises groupswhich, once activated, react specifically with other functional groups,for example amine or amide.

The term “spacer” relates to a moiety of several atoms within amolecule, which itself is free of reactive functions and spatiallyseparates two functional groups of the molecule. The spacer is acovalently bonded chain or ring structure consisting of carbon,phosphorus, sulphur, silicon, nitrogen and/or oxygen atoms. The spacermay contain substituting groups which do not contribute to the distancebetween the functional groups to be separated.

The term “group” relates to a moiety of several atoms within a molecule.Typically, these atoms form functional units such as a spacer, areactive function or a molecular structure that exerts a mesomeric orinductive effect.

The terms “functional linker” or “linker” relate to a functional groupthat connects two functional units within a molecule. The linker iscovalently bound to the functional groups.

The terms “linker”, “linker molecule”, “linker system” and “capturecompound” relate to a molecule that connects two other molecules byforming a covalent bond to each of the other molecule. The covalentbonds to the functional groups of the two other molecules only occurunder certain reaction conditions. In particular, the terms “linker”,“linker molecule”, “linker system” and “capture compound” relate tocompounds which fall under formula (1) and can generate a connectionbetween an N-terminus of a peptide and a solid support.

The term “substituted” relates to the addition of an atom or a moleculargroup or compound to a parent compound. The substituent group orcompound can be added protected or unprotected to one or more availablepositions in the parent molecule. The substituent group or compounditself may be substituted or unsubstituted and bound directly or througha linking group or moiety such as an alkyl, amide or hydrocarbonyl groupto the parent molecule. Substituting groups or compounds include, forexample, halogens, oxygen, nitrogen, sulfur, hydroxyl, alkyl, alkenyl,alkynyl, carboxyl (—C(O)OR^(a)), acyl (—C(O)R^(a)) groups, aliphatic,alicyclic groups, alkoxy, amino ((—N(R^(b))(R^(c))), imino (═NR^(b)),amido (—C(O)N(R^(b))(R^(c)) or —N(R^(b))C(O)R^(a)) groups, hydrazinederivatives (—C(NH)NR^(a)R^(b)), triazoles, tetrazoles (CN₄H₂), azido(—N₃), nitro (—NO₂), cyano (—CN), isocyano (—NC), cyanato (—OCN),isocyanato (—NCO), thiocyanato (—SCN); isothio-cyanato (—NCS); carbamido(—OC(O)N(R^(b))(R^(c)) or —(R^(b))C(O)OR^(a)) groups, thiols (—SR^(b)),sulfinyl (—S(O)R^(b)), sulfonyl (—S(O)₂R^(b)), sulfonamidyl(—S(O)₂N(R^(b))(R^(c)) or —N(R^(b))S(O)₂R^(b)) groups and fluoriniertefluorinated moieties such as —CF₃, —OCF₃, —SCF₃, —SOCF₃ or —SO₂CF₃.R^(a), R^(b) and R^(c) is independently from each other H or a furthersubstituting group.

The term “alkyl” relates to a saturated straight or branched hydrocarbonchain with up to 12 carbon atoms. Examples of preferred alkyl groups aremethyl, ethyl, propyl, butyl, isopropyl, n-hexyl, octyl, decyl groups.

The term “aryl” relates to a hydrocarbon moiety with alternating doubleand single bonds between the carbon atoms, wherein a ring structure isformed.

The term “leaving group” relates to a functional group within a moleculethat exerts a -M and/or —I effect and can thus easily be cleaved off,wherein the binding electron pair remains with the leaving group aftercleavage.

The term “surface-modified solid support” relates to a solid structuresuch as sepharose, agarose or cellulose units, silica gel orpolydextrans modified by synthetic or natural polymers such aspolysaccharides, polylysine, polyarylamide, polyethylene glycol (PEG) oracrylamide-PEG copolymers. The surface of the solid support ischaracterized by aldehyde, ketone, hydroxylamine or hydrazine groups.

In some embodiments, Z is an electron-withdrawing leaving group whichexerts a -M and/or —I effect and, in the case of a heterolytic bondcleavage, keeps the binding electron pair.

In some embodiments, Z is an electron-withdrawing leaving group, whereinthe acid corresponding to the anion of the leaving group ischaracterized by a pks value of less than five.

In some embodiments, Z is an electron-withdrawing leaving group, whereinthe acid corresponding to the anion of the leaving group ischaracterized by a pks value of less than five, and wherein the leavinggroup in particular exerts a -M and/or —I effect, and in the case of aheterolytic bond cleavage keeps the binding electron pair.

In some embodiments, U is the cleavage activating part of the functionallinker, wherein the activating part is a group which allows anionformation by -M and —I effects, and stabilizes the resulting anioniccompound by an electron pair shift, wherein this stabilization leads toa heterolytic bond cleavage between U and X₂.

In some embodiments, B is selected from Boc (—C═OOtBu), trityl(—C(Ph)₃), Mmt (—C(Ph)₂C₆H₄OMe), DMT (—C(Ph)(C₆H₄OMe)₂), Cbz(—C═OOCH₂Ph), benzylideneamine (═CPh), phthalimides (═(CO)₂C₆H₄),p-toluenesulfonamides (—SO₂C₆H₄Me), benzylamine (—CH₂Ph), acetamides(—COMe), trifluoroacetamide (—COCF₃), Dde(1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-ethyl) and1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl (ivDde),wherein particularly B is Boc.

In some embodiments, the acetal- or ketal protecting groups are selectedfrom

wherein r is 0 to 12, in particular 0, 1 or 2.

The skilled person is aware that the inventive compound can also beformed with other acid labile protecting groups.

In some embodiments, T is selected from substituted or unsubstitutedC₁-C₁₂-alkyl, in particular C₁-C₆-alkyl, in particular C₁-C₃-alkyl,—R⁵—C(═O)—NH—R⁶—, —R⁵—C(═O)—O—R⁶—, —R⁵—C(═O)—O—, —C(═O)—O—R⁶—,—C(═O)—NH—R⁶—, —C(═O)—, —C(═O)—O—, wherein R⁵ and R⁶ are independentlyfrom each other selected substituted or unsubstituted C₁-C₁₂-alkyls, inparticular C₁-C₆-alkyls, in particular C₁-C₃-alkyls.

When T is a substituted alkyl, the substituents are particularly thosethat increase water solubility, for example —SO₃H, —CO₂H or —NO₂.

In some embodiments, T is selected from —CH₂—, —CH₂—C(═O)—NH—(CH₂)₂—,—(CH₂)—C(═O)—O—(CH₂)₂—, —CH₂—C(═O)—O—, —C(═O)—O—, —C(═O)—O—(CH₂)₂—, and—C(═O)—.

In some embodiments, T is selected from —CH₂—, —CH₂—C(═O)—NH—(CH₂)₂—,—C(═O)—O—(CH₂)₂—, —CH₂—C(═O)—O—, and —C(═O)—.

In some embodiments, U of the moiety T-U—Y is selected from the moietiesaccording to the formulas (5), (6), (7), (8), (9), (10) and (11), inparticular from (5), (6), (8), (9) and (10),

wherein R⁸ is selected from C₁-C₆-alkyl, CF₃, CH₂CF₃,

in particular from Boc-Lys(Boc)-,

-   -   wherein R⁷ _(n), R⁹ _(m), R¹⁰ _(p), R¹¹ _(q), R¹³ _(r) and R¹⁴        _(s) is selected from C₁-C₆-alkyl or —I and/or -M-effects        generating substituents, in particular C₁-C₃-alkyls, —F, —Cl,        —Br, —I, —CN —NO₂, —N₃, —CF₃, —SO₃H, —CO₂H,    -   wherein n equals 0, 1, 2, 3 or 4, in particular n is 0 oder 1,        in particular 0,    -   wherein m equals 0, 1, 2 or 3, in particular m is 0 oder 1, in        particular 0,    -   wherein p equals 0, 1, 2, 3 or 4, in particular p is 0 oder 1,        in particular 0,    -   wherein q equals 0, 1, 2 or 3, in particular q is 0 oder 1, in        particular 0,    -   wherein r equals 0, 1, 2 or 3, in particular r is 0 oder 1, in        particular 0,    -   wherein s equals 0, 1, 2 or 3, in particular s is 0 oder 1, in        particular 0.

In some embodiments, R⁷ _(n), R⁹ _(m), R¹⁰ _(p), R¹¹ _(q), R¹³ _(r) andR¹⁴ _(s) is selected from C₁-C₆-alkyl or —I and/or -M-effects generatingsubstituents, in particular C₁-C₃-alkyls, —F, —Cl, —Br, —I, —CN —NO₂,—N₃, —CF₃, —SO₃H, and —CO₂H, in particular —F, —Cl, —Br, —I, —NO₂, and—N₃

-   -   wherein n equals 0, 1, 2, 3 or 4, in particular n is 0 oder 1,        in particular 0,    -   wherein m equals 0, 1, 2 or 3, in particular m is 0 oder 1, in        particular 0,    -   wherein p equals 0, 1, 2, 3 or 4, in particular p is 0 oder 1,        in particular 0,    -   wherein q equals 0, 1, 2 or 3, in particular q is 0 oder 1, in        particular 0,    -   wherein r equals 0, 1, 2 or 3, in particular r is 0 oder 1, in        particular 0,    -   wherein s equals 0, 1, 2 or 3, in particular s is 0 oder 1, in        particular 0.

In some embodiments, R⁷ _(n), R⁹ _(m), R¹⁰ _(p), R¹¹ _(q), R¹³ _(r) andR¹⁴ _(s) is selected from substituents that increase water solubility,in particular from —NO₂, —SO₃H and —CO₂H.

In some embodiments, Z is selected from the group —F, —Cl, —Br, —I, —N₃,—SR¹², —OCF₃, —OCH₂CF₃, —OSO₂CF₃, —SO₂C₆H₄CH₃, —SO₂CF₃, —SO₂CH₃

wherein R¹² is a C₁-C₆-alkyl-, an aryl- or a benzyl residue.

In some embodiments, Z is selected from —Cl,

in particular from

In some embodiments, X₁ is a moiety of formula (2) or (3), wherein R³ isH, R¹ and R² comprise a Boc protecting group or R¹ is H and R² is a Bocprotecting group.

In some embodiments, X₁ is a moiety of formula (3), wherein R¹ and R³ isH and R² is a Boc protecting group.

In some embodiments, Y is of the form —O—C(═O)—.

In some embodiments, T is of the form —CH₂—C(═O)—NH—(CH₂)₂—,—CH₂—C(═O)—O—(CH₂)₂—, —C(═O)—O—(CH₂)₂— and U is a moiety of formula (5)or (6),

In some embodiments, T is of the form —C(═O)—O—(CH₂)₂— or—CH₂—C(═O)—NH—(CH₂)₂— and U is a moiety of formula (6).

In some embodiments, T is of the form —C(═O)—O—(CH₂)₂— or—CH₂—C(═O)—NH—(CH₂)₂— and U is a moiety of formula (6).

In some embodiments, T is of the form —CH₂—C(═O)—NH—(CH₂)₂— and U is amoiety of formula (6).

In some embodiments, T is of the form —CH₂—C(═O)—O— or —C(═O)—O— and Uis a moiety of formula (7),

wherein R⁷ is selected from C₁-C₆-Alkyl or —I and/or -M-effectgenerating substituents, in particular C₁-C₃-alkyl, —F, —Cl, —Br, —I,—CN —NO₂, —N₃, —CF₃, —SO₃H, and —CO₂H, wherein n equals 0, 1, 2, 3 or 4,in particular 0 or 1, in particular 0.

In some embodiments, T is of the form —CH₂—C(═O)—O—, and U is a moietyof formula (7), wherein n equals 0.

In some embodiments, T is of the form —CH₂—, and U is a moiety offormula (8),

wherein R⁸ is Boc-Lys(Boc)- and and r equals 0.

In some embodiments, T is of the form —CH₂— or —(C═O)—, and U is amoiety of formula (9),

wherein s equals 0.

In some embodiments, T is of the form —CH₂—, and U is a moiety offormula (9).

In some embodiments, T is of the form —C(═O)—, and U is a moiety offormula (10),

wherein m equals 0, Y is of the form —SO₂— and Z is Cl.

In some embodiments, the compound of formula (1) is selected from2,2-dimethylpropanoyloxy-[2-[2-[2-(4-nitrophenoxy)carbonyloxypropylsulfonyl]ethylamino]-2-oxo-ethoxy]amino]2,2-dimethylpropanoate (formula (14)), [[2-[2-[2-(4-nitrophenoxy)carbonyloxypropylsulfonyl]ethylamino]-2-oxoethoxy]amino]2,2-dimethylpropanoate (formula (15)),[2-(4-chlorosulfonyl-3-nitrobenzoyl)hydrazino] 2,2-dimethylpropanoate(formula (16)),[2,2-dimethylpropanoyloxy-[2-[4-1[(2,5-dioxopyrrolidine-1-yl)oxycarbonyloxymethyl]phenoxy]-2-oxo-ethoxy]amino]2,2-dimethylpropanoate (formula (17)),[2-(2,2-dimethylpropanoyloxy)-2-[2-[2-[2-(2,5-dioxopyrrolidine-1-yl)oxycarbonyloxypropylsulfonyl]ethylamino]-2-oxo-ethyl]hydrazino]2,2-dimethylpropanoate (formula (18)),[2-(2,2-dimethylpropanoyloxy)-2-[2-[2-[2-(2,5-dioxopyrrolidine-1-yl)oxycarbonyloxyethylsulfonyl]ethylamino]-2-oxo-ethyl]hydrazino]2,2-dimethylpropanoate (formula (19)),[2-[5-azido-2-[(2,5-dioxopyrrolidine-1-yl)oxycarbonyloxymethyl]benzoyl]hydrazino]2,2-dimethylpropanoate(formula (20)),[3-[(2,2-dimethylpropanoyloxyamino)carbamoyl]-4-[(2,5-dioxopyrrolidine-1-yl)oxycarbonyloxymethyl]phenyl]2,6-bis(2,2-dimethylpropanoyloxyamino) hexanoate (formula (21)),[2-[2-[2-[2-(4-nitrophenoxy)carbonyloxypropylsulfonyl]ethylamino]-2-oxo-ethyl]hydrazino]2,2-dimethylpropanoate (formula (22)),[2-[2-[2-[2-(2,5-dioxopyrrolidine-1-yl)oxycarbonyloxy-propylsulfonyl]ethylamino]-2-oxo-ethyl]hydrazino]2,2-dimethylpropanoate (formula (23)),[2-[2-[4-[(2,5-dioxopyrrolidine-1-yl)oxycarbonyloxymethyl]phenoxy]-2-oxo-ethyl]hydrazino]2,2-dimethyl-propanoate (formula (24)),[2-[2-[2-(4-nitrophenoxy)carbonyloxyethylsulfonyl] ethoxycarbonyl]hydrazino] 2,2-dimethylpropanoate (formula (25)),[3-[[2-(2,2-dimethylpropanoyloxy)hydrazino]methyl]-4-[(2,5-dioxopyrrolidine-1-yl)oxycarbonyloxymethyl]phenyl] 2,6-bis(2,2-dimethylpropanoyloxyamino)hexanoate (formula (26)),[2-[[5-azido-2-[(2,5-dioxopyrrolidine-1-yl)oxycarbonyloxymethyl]phenyl]methyl]hydrazino]2,2-dimethylpropanoate (formula (27)),

In some embodiments, the compound of formula (1) is selected from acompound of formula (14), (15), (16), (17), (18), (19), (20) and (21).

In some embodiments, the compound of formula (1) is selected from acompound of formula (16), (19), (20) and (21).

In a further aspect, the object of the invention is achieved by the useof a compound according to the first aspect that forms a connectionbetween the N-terminal amino group of a full-length peptide and a solidphase.

In a further aspect, the object of the invention is achieved by acompound of formula (12), X₁-L-Y-PEP (12), wherein X₁, L and Y aredefined according to the first aspect and its embodiments, and whereinPEP comprises a full-length peptide that is bound to X₂′ via itsN-terminus.

In a further aspect, the object of the invention is achieved by acompound of formula (13), D-X₁′-L-Y-PEP (13), wherein D is asurface-modified solid support, which is characterized in that thesurface is modified by synthetic or natural polymers, wherein X₁′ is ofthe form —NH—O—, —NH—NH— or —C(═O)— and wherein L, Y and PEP are definedaccording to the first aspect and its embodiments.

In some embodiments, the surface-modified solid support D ischaracterized by modified polysaccharides.

In some embodiments, the surface-modified solid support D ischaracterized by aldehyde- or hydrazine-modified sepharose/agarose orcellulose.

In a further aspect, the object of the invention is achieved by a methodfor the purification of peptides, in particular of peptides prepared bysolid phase peptide synthesis (SPPS), comprising the following steps:

-   -   i. contacting a composition of a full-length peptide to be        purified and at least one impurity, in particular at least one        acetylated truncated sequence, with a capture compound that is        defined according to the first aspect and its embodiments, and        subsequent reaction to a compound of formula (12),    -   ii. cleavage of the acid labile protecting groups by addition of        an acid,    -   iii. contacting the composition of ii. with a surface-modified        solid support, wherein a covalent hydrazone or oxime bond is        formed between the capture compound and the solid support, and a        compound of formula (13) is provided,    -   iv. cleavage of the full-length peptide from the solid support.

In some embodiments, the method comprises an optional modification stepiiia (cyclization) that is performed after step iii.

In some embodiments, step i. comprises contacting a mixture offull-length peptide and truncated sequences, that are still at the solidphase (the synthesis resin), with a compound (capture molecule) of thegeneral formula X₁-L-X₂ wherein X₁, X₂ and L are as defined above andwherein the step of contacting leads to a reaction of the compoundX₁-L-X₂ at X₂ with the free N-terminal amino group of the full-lengthpeptide to form a covalent bond. Cleavage of the peptides from the solidphase (synthesis resin) is performed by means of acids, whereby amixture of full-length peptide covalently bound to the capture moleculeand acetylated truncated sequences of peptides from a solid phasepeptide synthesis (SPPS) is obtained. Separation of the solid and liquidphase is performed for example by filtration. The non-peptide impuritiesare removed by precipitation in ether at a temperature of −78° C. to 0°C. Preferably, the acid mixture is added to the provided ether, whereinall peptide material precipitates and organic impurities remain in theether. The etheric solution is then separated from the peptide mixture,e.g. by centrifugation. The peptide mixture is obtained as an amorphoussolid.

In some embodiments, step ii. comprises dissolution of the amorphoussolid from step i) in an at least partially aqueous buffer solution at apH value between 2 and 4, preferably between 2.5 and 3.5, particularlypreferably at 3. The pH is adjusted by adding suitable acids or bases.

In some embodiments, step ii) comprises dissolution of the amorphoussolid from step i) in an at least partially aqueous buffer solution at apH value between 2 and 4, preferably between 2.5 and 3.5, particularlypreferably at 3, DMSO or hexafluoroisopropanol.

DMSO or hexafluoroisopropanol may be used as solvents. When DMSO isused, usually a buffer such as sodium citrate buffer with guanidiniumchloride is subsequently added.

In some embodiments, step ii) comprises first dissolution of theamorphous solid from step i) in DMSO and then adding an at leastpartially aqueous buffer solution at a pH value between 2 and 4,preferably between 2.5 and 3.5, particularly preferably at 3, or stepii) comprises dissolution of the amorphous solid from step i) inhexafluoroisopropanol.

In some embodiments the at least partially aqueous buffer is a sodiumcitrate buffer with guanidinium chloride, particularly sodium citratepuffer (pH 3.5) with 7 M guanidinium chloride.

In some embodiments, step ii) comprises first dissolution of theamorphous solid from step i) in DMSO or hexafluoroisopropanol and thenadding sodium citrate buffer with guanidinium chloride, particularlysodium citrate puffer (pH 3.5) with 7 M guanidinium chloride.

In some embodiments, step iii. comprises contacting the mixture from ii)with a surface-modified solid support (purification resin) to covalentlybind the full-length peptides, that are modified with the capturemolecule (step i), by forming a hydrazone or oxime bond. The addition ofamines and/or acetic acid as a catalyst to improve the kinetics of thebinding reaction is particularly advantageous here.

The truncated peptide sequences not bound to the solid support via thehydrazone/oxime bond are removed by washing with organic solvents and/orwith water and aqueous buffer solution, preferably with the addition ofchaotropic substances, in order to dissolve peptides which may not becovalently bound.

After step iii, an optional step iiia may be performed to modify thepeptide by intramolecular cyclization via disulfide formation. In someembodiments, cyclization of the moiety PEP of the compound of formula(13) is performed by oxidation of two or more residues bearing anucleophilic thiol towards a disulfide bridge.

In some embodiments, step iv. comprises separating the full-lengthpeptides from the solid phase by cleaving the linker L from Y underbasic (nucleophilic) conditions, wherein Y is released in the form ofCO₂ or SO₂.

Upon cleavage of the full-length peptide starting from the formula (13)(D-X1′-L-Y-PEP), the full-length peptide (PEP), CO₂ or SO₂ (Y′) andD-X1′-L or D′ and X₁′-L′ are formed.

In some embodiments, the solid support comprises on its surface thefunctional groups aldehyde, ketone, hydroxylamine and hydrazine.

In some embodiments, the solid support comprises on its surface thefunctional group —O—CH₂—CHO.

In some embodiments, the solid support comprises on its surface thefunctional groups —ONH₂ or —N₂H₃.

In some embodiments, the solid phase is separated from the desiredfull-length peptides by filtration; the solid phase (purification resin,D) is regenerated by treatment with hydrazine (H₄N₂) and/or ammoniumhydroxide H₄NOH and/or aldehydes and/or ketones and/or washing withwater.

For cleaving the peptides form the synthesis resin (step i), acids areused, organic and inorganic acids with a pks value below 4 are preferredhere. Acids selected from the group of acids containing fluorine areparticularly suitable: trifluoroacetic acid (TFA), hydrofluoric acid(HF) and trifluoromethanesulfonic acid. Hydrobromic acid (HBr),hydrochloric acid (HCl), sulphurous acid (H₂SO₃), sulphuric acid(H₂SO₄), phosphoric acid (H₃PO₄), nitric acid (HNO₃) or methanesulfonicacid are also suitable.

For the precipitation in step i) organic solvents are used which are inliquid state at the precipitation temperatures; such solvents aregenerally known to the skilled person. Organic solvents from the groupof ethers are preferred, particularly diethyl ethers and/or methyltert-butyl ethers are preferred. Alkanes which are in liquid state atthe precipitation temperatures can also be used, wherein n-hexane and orn-pentane are particularly preferred. Suitable, at least partiallyaqueous buffer solutions which are used in step ii) are known to theskilled person, namely buffers which have a buffer capacity in the pHrange 2-5, thus buffers with the anions: citrate, malate, format,lactate, succinate, acetate, pivalate, and phosphate in combination withthe cations: sodium, potassium, ammonium (NH₄, NMe₄, NEt₄, NPr₄, NBu₄,HNC₅H₅).

Organic or inorganic acids, preferably HCl and as bases preferablyalkali metal and/or alkaline earth metal hydroxides, particularlypreferably NaOH and/or KOH, can be used for adjusting the pH value.

For better solubility of the peptide it may be advantageous in step ii)to add water-miscible organic solvents to the system, such solvents aregenerally known to the skilled person and may be selected from thegroup: dimethylformamide (DMF), acetonitrile, tetrahydrofuran (THF),dioxane, pyridine, acetone, dimethyl sulfoxide (DMSO), methanol,ethanol, 1-propanol, 2-propanol, 1-butanol, formamide,N-methylpyrolidone (NMP).

Amines and/or acetic acid may be added to the aqueous solution toaccelerate immobilization in step iii). Amines may be selected from thegroup consisting of: pyridine, piperidine, methylamine, ethylamine,propylamine, butylamine, aniline and dimethylamine.

Synthetic and natural polymers can be used as surface-modified solidsupport (purification resin) in step iii). The surface modification issuch that it reacts with X₁ to hydrazones or oximes. If X₁ is a moietyaccording to formula (2) or (3), the surface modification consists ofaldehyde or ketone groups, which then react accordingly to hydrazones oroximes. If X₁ is an aldehyde or ketone function of the general formula(4), the surface modification should comprise —ONH₂, or —N₂H₃.Surface-modified natural as well as biopolymers are preferred as solidsupports, particularly preferably surface-modified polysaccharides. Mostpreferred is the use of aldehyde-modified sepharose/agarose andcellulose, wherein X₁ is a moiety of formula (3), wherein R¹ and R³ areH.

In step iii (washing of the full-length peptides bound to thepurification resin) can be washed with water, aqueous washing solutionsor organic solvents. Suitable chaotropic substances to the aqueouswashing solution in step iii. are: barium salts, guadiniumhydrochloride, guadinium thiocyanates, thiocyanates, perchlorates,iodides, butanol, phenol, thiourea, urea, or ammonium sulfate. Solventsmay be selected from the group of: dichloromethane (DCM),trichloromethane, carbon tetrachloride, ethyl acetate, diethyl ether,methyl tert-butyl ether, acetic acid, 2,2,2-trifluoroethanol,hexafluoroisopropanol, dimethylformamide (DMF), acetonitrile.Tetrahydrofuran (THF), dioxane, pyridine, acetone, dimethyl sulfoxide(DMSO), methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, phenol,formamide and N-methylpyrolidone (NMP).

The peptide can optionally be modified in step iiia by intramoleculardisulfide formation. Cyclization is often used to improve in vivopeptide stability and potency: Cyclic peptides are highly resistant toexo- and endoproteases. Due to their more rigid conformation, goodbinding affinities to desired targets are observed. Cyclization may beachieved by intramolecular disulfide formation. For this, functionalgroups of the peptide, such as thiol moieties, are utilized.

Cyclization may also be achieved by intramolecular disulfide bondformation. For example, the thiol moieties of two cysteine residues inthe peptide may form a disulfide.

In certain embodiments, the cyclization is performed in the presence ofair or in the presence of an oxidative additive.

In certain embodiments, the nucleophilic thiols are oxidized by a basicaqueous solution with a pH >7, particularly pH >7 to pH 8.5, in themodification step to yield macrocyclic bridged peptides.

In certain embodiments, the nucleophilic thiols are oxidized by a basicaqueous solution with a pH >7, particularly pH >7 to pH 8.5, and/or inthe presence of air (particularly in the presence of oxygen), and/or orin the presence of an oxidative additive, in particular selected fromDMSO, iodine, N-chlorosuccinimide, Tl(OAc)₃, Tl(CF₃COO)₃,CH₃SiCI₃-Ph(SO)Ph, [Pt(ethylenediamine)₂Cl₂]Cl₂,2,2′-Dithiobis(5-nitropyridine), 5,5′-dithiobis-(2-nitrobenzoic acid),trans-[Pt—(CN)₄Cl₂]²⁻, glutathione-glutathione disulfide, K₃Fe(CN)₆.

The oxidative additives may be used as such or solved in a suitablesolvent. The oxidation may be performed in organic, organic-aqueous,acidic or basic solutions.

In certain embodiments, the nucleophilic thiols are oxidized in thepresence of air (particularly in the presence of oxygen), or in thepresence of an oxidative additive, in particular selected from DMSO,iodine, N-chlorosuccinimide, —Tl(OAc)₃, —Tl(CF₃COO)₃,—CH₃SiCI₃-Ph(SO)Ph, [Pt(ethylenediamine)₂Cl₂]Cl₂,2,2′-Dithiobis(5-nitropyridine), 5,5′-dithiobis-(2-nitrobenzoic acid),trans-[Pt—(CN)₄Cl₂]²⁻, glutathione-glutathione disulfide, K₃Fe(CN)₆.

In certain embodiments, the nucleophilic thiols are oxidized in thepresence of DMSO, iodine, N-chlorosuccinimide, Tl(OAc)₃,[Pt(ethylenediamine)₂Cl₂]Cl₂, trans-[Pt—(CN)₄Cl₂]²⁻, glutathione.

In certain embodiments, the nucleophilic thiols are oxidized in thepresence of air (oxygen), DMSO, iodine, N-chlorosuccinimide, —Tl(OAc)₃,—Tl(CF₃COO)₃, —CH₃SiCI₃-Ph(SO)Ph, [Pt(ethylenediamine)₂Cl₂]Cl₂,2,2′-Dithiobis(5-nitropyridine), 5,5′-dithiobis-(2-nitrobenzoic acid),trans-[Pt—(CN)₄Cl_(2]) ²⁻, glutathione-glutathione disulfide, K₃Fe(CN)₆.

In certain embodiments, the nucleophilic thiols are oxidized in thepresence of DMSO, iodine, N-chlorosuccinimide, Tl(OAc)₃,[Pt(ethylenediamine)₂Cl₂]Cl₂, trans-[Pt—(CN)₄Cl_(2]) ²⁻, glutathione.

In certain embodiments, the nucleophilic thiols are oxidized in thepresence of N-Chlorosuccinimide, DMSO and glutathione-glutathionedisulfide.

In certain embodiments, the nucleophilic thiols are oxidized in thepresence of N-Chlorosuccinimide, DMSO and glutathione.

In certain embodiments, the peptide comprises at least two amino acidscomprising a nucleophilic thiol.

In certain embodiments, the peptide comprises 2 to 10, particularly 2 to8, even more particularly 2 to 6 amino acids comprising a nucleophilicthiol.

In certain embodiments, the peptide comprises 2 amino acids comprising anucleophilic thiol.

In certain embodiments, the peptide comprises at least two amino acidsindependently selected from cysteine, homocysteine or penicillamine.

In certain embodiments, the peptide comprises 2 to 10, particularly 2 to8, even more particularly 2 to 6 amino acids independently selected fromcysteine, homocysteine or penicillamine.

In certain embodiments, the peptide comprises 2 amino acidsindependently selected from cysteine, homocysteine or penicillamine.

The cleavage in step iv. is performed by bases in aqueous solutions ororganic solvents which dissolve peptides. Bases may be selected from thegroup of: LiOH, NaOH, KOH, ammonium (NH₄, NMe₄, NEt₄, NPr₄, NBu₄,HNEt(iPr)₂, HNMe₃, HNEt₃, HNPr₃, HNBu₃, HNC₅H₅) hydroxides, piperidine,methylamine, ethylamine, propylamine, butylamine, hydrazine,hydroxylamine, methylhydrazine and O-methylhydroxylamine. Organicsolvents which dissolve peptides may be selected from the group of:dimethylformamide (DMF), acetonitrile. Tetrahydrofuran (THF), dioxane,pyridine, acetone, dimethyl sulfoxide (DMSO), methanol, ethanol,1-propanol, 2-propanol and 1-butanol.

In certain embodiments, bases in step iv) are selected from the groupof: LiOH, NaOH, KOH, ammonium (NH₃, NMe₃, NEt₃, NPr₃, NBu₃, NEt(iPr)₂,HNMe₂, HNEt₂, HNPr₂, HNBu₂, HNC₅H₅) hydroxides, piperidine,ethanolamine, methylamine, ethylamine, propylamine, butylamine,hydrazine, hydroxylamine, methylhydrazine and O-methylhydroxylamine.

Filtration in step iv) is preferably performed using commerciallyavailable syringe reactors or filter systems. The filter pore sizesshould be between 10 and 100 μm.

In some embodiments, after or during cleavage of the full-length peptidefrom the solid support, the solid support D is cleaved from the residueX₁-L of the capture compound and the solid support is regenerated.

A particular advantage of the method according to the invention is thereversibility of the hydrazone and oxime bond.

The method described herein can be used like affinity chromatography dueto the equilibrium nature of the hydrazone/oxime bond. After washing outor away the impurities and cleaving the base-labile linker and thusobtaining the target peptide, the purification resin can be regeneratedagain and is thus accessible for further purification. If there areoriginally aldehyde or keto groups on the surface of the purificationresin, washing with acidic aqueous solution in which aldehydes orketones are dissolved restores the aldehyde or ketone function. If thereare originally hydrazine or hydroxylamine derivatives on the surface ofthe purification resin, washing with acidic aqueous solution withhydrazine or hydroxylamine restores the hydrazine or hydroxylaminefunction. The same material is used as for protein purification byaffinity chromatography, sepharose/agarose.

Furthermore, the method can be applied on cellulose, which is the mostcommon biomaterial on earth and therefore available at low cost.

Proteins are normally purified by affinity chromatography, and thismethod is also very cost-effective, efficient and scalable compared toHPLC purification. Due to the low pressures and higher loadingdensities, affinity chromatography is also much better suited for largesynthesis quantities than HPLC.

In a subaspect of the first aspect of the present invention, the objectis achieved by a compound which establishes as a linker a connectionbetween the N-terminal amino group of a full-length peptide and a solidphase. The compound according to the invention is of the general formula

X₁-L-X₂,

wherein

X₁ is selected from

wherein Y₁=O, N and wherein R₁ and R₂ may be the same or different andR₁ and R₂ is H or B, wherein B is a non-base-labile protecting group foran amino group which provides amines under acidic conditions.

In a preferred embodiment, B is selected from the group: Boc (—C═OOtBu),trityl (—C(Ph)₃), Mmt (—C(Ph)₂C₆H₄OMe), DMT (—C(Ph)(C₆H₄OMe)₂), Cbz(—C═OOCH₂Ph), benzylidenamine (═CPh), phthalimide (═(CO)₂C₆H₄),p-toluenesulfonamide (—SO₂ C₆H₄Me), benzylamine (—CH₂Ph), acetamide(—COMe), trifluoroacetamide (—COCF₃), Dde(1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-ethyl) and1-(4,4-dimethyl-2,6-dioxocyclohex-lylidene)-3-methylbutyl (ivDde).

The skilled person is also familiar with protecting groups from P. G. M.Wuts, T. W. Greene, Greene's Protective Groups in Organic Synthesis, 4thed., Wiley, 2007, pages 696-926.

X₁ may alternatively be

wherein R₃=H, or R₃ is a saturated or unsaturated, branched orunbranched, substituted or unsubstituted aliphatic or aromatic chainhaving a length of up to 12 carbon atoms, and wherein the aldehyde orketo group may be protected in a manner known to the skilled person

wherein n may be between 0 and 12, particularly preferably 0, 1 or 2.

All acetal-ketal protecting groups that can be cleaved under acidicconditions can serve as protected aldehyde or ketone. An overview can befound in particular in: P. G. M. Wuts, T. W. Greene, Greene's ProtectiveGroups in Organic Synthesis, 4th ed., Wiley, 2007, page 435-477.

X₂ may be

wherein Z is an electron-withdrawing protecting group which keeps thebinding electron pair in the case of a heterolytic bond cleavage. In aparticular embodiment, Z is selected from the group F, Cl, Br, J, N₃,SR₈ (wherein R₈ is defined as R₃), OCF₃, OCH₂CF₃,

X₂ thus represents a carbamate precursor which is to form a carbamatewith the amino group of the full-length peptide.

L is a functional linker that separates X₁ and X₂ and is cleavable underbasic conditions (nucleophilic). A selection of nucleophilicallycleavable linkers can be found in F. Guillier, D. Drain, M. Bradley,Linkers and Cleavage Strategies in Solid-Phase Organic Synthesis andCombinatorial Chemistry, Chem. Rev. 2000, 100, 2091-2157.

In some embodiments, the compounds of the general formula X₁-L-X₂ arepharmaceutically acceptable salts thereof.

In some embodiments, the compound of the general formula X₁-L-X₂ is usedas a linker which forms a connection between the N-terminal amino groupof a full-length peptide and a solid phase.

In a preferred embodiment, the linker L is selected from the generalstructures a, b or c

A: C₀-C₁₂, aromatic aliphatic, unsaturated, saturated

R₄: H, alkyl or electron-withdrawing group

R₅, R₆: identical or different H or alkyl C₁-C₁₂

R₇: identical or different H, electron-withdrawing group, or alkylC₁-C₁₂

wherein A is a saturated or unsaturated, branched or unbranched,substituted or unsubstituted aliphatic or aromatic chain having a lengthof 0 to 12 carbon atoms and wherein R₄=H, an alkyl chain of 0 to 12carbon atoms or a group capable of attracting electrons by an inductiveor mesomeric effect.

Wherein R₅ and R₆ may be the same or different and R₅, R₆=H, an alkylchain having a length of 1 to 12 carbon atoms or a group capable ofattracting electrons by an inductive or mesomeric effect. R₇ is H, analkyl chain having a length of 1 to 12 carbon atoms or a group capableof attracting electrons by an inductive or mesomeric effect.

R₈ is as R₃.

In another aspect, the object of the invention is achieved by a methodthat comprises the following steps.

i) Contacting a mixture of full length peptide and truncated sequences,that are still at the solid phase (the synthesis resin), with a compound(capture molecule) of the general formula X₁-L-X₂ wherein X₁, X₂ and Lare as defined above and wherein the step of contacting leads to areaction of the compound X₁-L-X₂ at X₂ with the free N-terminal aminogroup of the full-length peptide to form a covalent bond.

ii) Cleavage of the peptides from the solid phase (synthesis resin) bymeans of acids and obtaining a mixture of full-length peptide covalentlybound to the capture molecule and acetylated truncated sequences ofpeptides from a solid phase peptide synthesis (SPPS);

iii) Separation of the solid and liquid phase, e.g. by filtration;

iv) Removal of non-peptide impurities by precipitation in ether at atemperature of −78° C. to 0° C. Preferably the acid mixture is added tothe provided ether, wherein all peptide material precipitates andorganic impurities remain in the ether. The etheric solution is thenseparated from the peptide mixture, e.g. by centrifugation. The peptidemixture is obtained as an amorphous solid;

v) Dissolution of the amorphous solid from step iv) in an at leastpartially aqueous buffer solution at a pH between 2 and 4, preferablybetween 2.5 and 3.5, particularly preferably of 3. The pH is adjusted byadding suitable acids or bases;

vi) Contacting the mixture from v) with a surface-modified solid support(purification resin) to covalently bind the full-length peptidesmodified with the capture molecule (step i) by forming a hydrazone oroxime bond. The addition of amines and/or acetic acid as a catalyst toimprove the kinetics of the binding reaction is particularlyadvantageous here;

vii) Removal of truncated peptide sequences not bound to the solidsupport via the hydrazone/oxime bond by washing with organic solventsand/or with water and aqueous buffer solution, preferably with theaddition of chaotropic substances, in order to dissolve peptides whichmay not be covalently bound;

viii) Separation of the full-length peptides from the solid phase bycleaving the linker L under basic (nucleophilic) conditions;

ix) Filtration to separate the solid phase from the desired full-lengthpeptides; and

x) Regeneration of the solid phase (purification resin) by treatmentwith hydrazine (H₄N₂) and/or ammonium hydroxide H₄NOH and/or aldehydesand/or ketones and/or washing with water.

For cleaving the peptides form the synthesis resin (step ii), acids areused, organic and inorganic acids with a pks value below 4 are preferredhere. Acids selected from the group of acids containing fluorine areparticularly suitable: trifluoroacetic acid (TFA), hydrofluoric acid(HF) and trifluoromethanesulfonic acid. Hydrobromic acid (HBr),hydrochloric acid (HCl), sulphurous acid (H₂SO₃), sulphuric acid(H₂SO₄), phosphoric acid (H₃PO₄), nitric acid (HNO₃) or methanesulfonicacid are also suitable.

For the precipitation in step iii) organic solvents are used which arein liquid state at the precipitation temperatures; such solvents aregenerally known to the skilled person. Organic solvents from the groupof ethers are preferred, particularly diethyl ethers and/or methyltert-butyl ethers are preferred. Alkanes which are in liquid state atthe precipitation temperatures can also be used, n-hexane and orn-pentane are particularly preferred.

Suitable, at least partially aqueous buffer solutions which are used instep v) are known to the skilled person, namely buffers which have abuffer capacity in the pH range 2-5, thus buffers with the anions:citrate, malate, format, lactate, succinate, acetate, pivalate, andphosphate in combination with the cations: sodium, potassium, ammonium(NH₄, NMe₄, NEt₄, NPr₄, NBu₄, HNC₅H₅).

Organic or inorganic acids, preferably HCl and as bases preferablyalkali metal and/or alkaline earth metal hydroxides, particularlypreferably NaOH and/or KOH, can be used for adjusting the pH value.

For better solubility of the peptide it may be advantageous in step v)to add water-miscible organic solvents to the system, such solvents aregenerally known to the skilled person and may be selected from thegroup: dimethylformamide (DMF), acetonitrile, tetrahydrofuran (THF),dioxane, pyridine, acetone, dimethyl sulfoxide (DMSO), methanol,ethanol, 1-propanol, 2-propanol, 1-butanol, formamide,N-methylpyrolidone (NMP).

Amines and/or acetic acid may be added to the aqueous solution toaccelerate immobilization in step vi). Amines may be selected from thegroup consisting of: pyridine, piperidine, methylamine, ethylamine,propylamine, butylamine, aniline and dimethylamine.

Synthetic and natural polymers can be used as surface-modified solidsupport (purification resin) in step vi). The surface modification issuch that it reacts with X₁ to hydrazones or oximes. If X₁=

(Y₁=NH, O) the surface modification consists of aldehyde or ketonegroups, which then react accordingly to hydrazones or oximes. If X₁ isan aldehyde or ketone function of the general formula —R₃C═O, thesurface modification should comprise a NH₂—Y₁—R₇ group (R₇=solidsupport). Surface-modified natural as well as biopolymers are preferredas solid supports, particularly preferably surface-modifiedpolysaccharides. Most preferred is the use of aldehyde-modifiedsepharose/agarose and cellulose and X₁=NH₂—NH—C═OO—.

In step vi (washing of the full-length peptides bound to thepurification resin) can be washed with water, aqueous washing solutionsor organic solvents. Suitable chaotropic substances to the aqueouswashing solution in step iii. are: barium salts, guadiniumhydrochloride, guadinium thiocyanates, thiocyanates, perchlorates,iodides, butanol, phenol, thiourea, urea, ammonium sulfate. Solvents maybe selected from the group: dichloromethane (DCM), trichloromethane,carbon tetrachloride, ethyl acetate, diethyl ether, methyl tert-butylether, acetic acid, 2,2,2-trifluoroethanol, hexafluoroisopropanol,dimethylformamide (DMF), acetonitrile. Tetrahydrofuran (THF), dioxane,pyridine, acetone, dimethyl sulfoxide (DMSO), methanol, ethanol,1-propanol, 2-propanol, 1-butanol, phenol, formamide andN-methylpyrolidone (NMP).

The cleavage in step vii. is performed by bases in aqueous solutions ororganic solvents which dissolve peptides. Bases may be selected from thegroup: LiOH, NaOH, KOH, ammonium (NH₄, NMe₄, NEt₄, NPr₄, NBu₄,HNEt(iPr)₂, HNMe₃, HNEt₃, HNPr₃, HNBu₃, HNC₅H₅) hydroxides, piperidine,methylamine, ethylamine, propylamine, butylamine, hydrazine,hydroxylamine, methylhydrazine and O-methylhydroxylamine. Organicsolvents which dissolve peptides may be selected from the group:dimethylformamide (DMF), acetonitrile. Tetrahydrofuran (THF), dioxane,pyridine, acetone, dimethyl sulfoxide (DMSO), methanol, ethanol,1-propanol, 2-propanol and 1-butanol. The filtration in step viii) ispreferably performed using commercially available syringe reactors orfilter systems. The filter pore sizes should be between 10 and 100 μm.

In some embodiments, the method according to the invention ischaracterized in that the solid and liquid phases are separated byfiltration.

In some embodiments, the method according to the invention ischaracterized in that in step vi. amines and/or acetic acid are added ascatalyst.

In some embodiments, the method according to the invention ischaracterized in that the buffer solution in step v. has a pH valuebetween 2.5 and 3.5 and preferably of 3.

In some embodiments, the method according to the invention ischaracterized in that the removal in step vii. is performed by washingwith organic solvents and/or with water and aqueous buffer solution,preferably with the addition of chaotropic substances, in order todissolve peptides which may not be covalently bound.

In some embodiments, the method according to the invention ischaracterized in that the separation of the full-length peptides fromthe solid phase in step viii. is performed by cleaving the linker Lunder basic (nucleophilic) conditions.

In some embodiments, the method according to the invention ischaracterized in that for the separation of the peptides from thesynthesis resin in step ii. acids with a pks value below 4 are used.

In some embodiments, the method according to the invention ischaracterized in that for precipitation in step iii. organic solventsfrom the group of ethers, particularly preferably diethyl ethers and/ormethyl tert-butyl ethers or n-hexane and or n-pentane are used.

In some embodiments, the method according to the invention ischaracterized in that synthetic and natural polymers are used assurface.-modified solid support (purification resin), e.g.surface-modified polysaccharides, particularly preferablyaldehyde-modified sepharose/agarose, or cellulose and X₁=NH₂—NH—C═OO—.

A particular advantage of the method according to the invention is thereversibility of the hydrazone and oxime bond.

The method described herein can be used like affinity chromatography dueto the equilibrium nature of the hydrazone/oxime bond. After washing outor away the impurities and cleaving the base-labile linker and thusobtaining the target peptide, the purification resin can be regeneratedagain and is thus accessible for further purification. If there areoriginally aldehyde or keto groups on the surface of the purificationresin, washing with acidic aqueous solution in which aldehydes orketones are dissolved restores the aldehyde or ketone function. If thereare originally hydrazine or hydroxylamine derivatives on the surface ofthe purification resin, washing with acidic aqueous solution withhydrazine or hydroxylamine restores the hydrazine or hydroxylaminefunction. The same material is used as for protein purification byaffinity chromatography, sepharose/agarose.

Furthermore, the method can be applied on cellulose, which is the mostcommon biomaterial on earth and therefore available at low cost.

Proteins are normally purified by affinity chromatography, and thismethod is also very cost-effective, efficient and scalable compared toHPLC purification. Due to the low pressures and higher loadingdensities, affinity chromatography is also much better suited for largesynthesis quantities than HPLC.

In the following, without limiting the generality of the teaching, theinvention will be explained by means of some examples with reference tothe figures.

General Synthesis Scheme

The capture molecules according to the invention can be preparedaccording to the general synthesis scheme (1). According to this scheme,the nucleophilic left part of the linker molecule can be varied betweenhydroxylamine and hydrazine. The cleavable part of the linker L can alsobe varied. A distinction can be made between sulfonlinkers and phenolester linker systems. In addition, the right carbonate part of thelinker system may have different leaving groups. This results in aplethora of possible combinations. The building blocks L1 and L3 arecommercially available. L2 can be prepared according to scheme 2.

L1, L2:

EXAMPLES Example 1. Demonstration of the Reversibility of theHydrazone/Oxime Bond

The reversible binding of the peptide to aldehyde-modified agarose beadsis demonstrated in the following using the example of hydrazone binding;due to the electronic similarity (see also A. Dirksen, P. Dawson,Bioconjugate Chem. 2008, 19, 2543-2548), the results are applicable tothe oxime bond. It is shown that the equilibrium can be controlled bythe addition of hydrazine (N₂H₄).

Peptide 3 was bound to the support 1 in the conjugation buffer (0.1 MNH₄OAc, 0.1 M PhNH₂, pH=3) in 30 min (FIG. 6: Scheme 3). Subsequently,it was washed with water and the supernatant removed. Then a solution of0.5 volume percent hydrazine hydrate was added to the beads and theabsorption was measured at 278 nm of the supernatant (200 μL) at a timeinterval (with the ND-I000 spectrophotometer from NanoDrop Technologie).It was found that after 10 min, 80% of the peptide can be measured inthe supernatant. After another 50 minutes, 86% of the peptide wasrecovered. A half-life of three minutes was determined by non-linearregression, FIG. 2 shows the adsorption at 278 nm when measuring thesupernatant after addition of N₂H₄.

Example 2: Purification of a Peptide after Native Chemical Ligation(NCL)

The purification of a peptide from a complex system containing a mixtureof peptide material as well as organic and inorganic impurities wasperformed.

The mixture to be purified was obtained after an NCL (FIG. 7: scheme 4).This reaction is performed in an aqueous buffer system and is used tosynthesize larger peptides and protein domains. After 24 h reaction timeof the NCL the raw mixture is obtained, which essentially contained thedesired ligation product and the thioester (H-YENRIK-MESA), which wasused in excess. This can be seen in the chromatogram (UPLC-MS fromWaters, Acquity H-Class PDA/QDa, on Polaris C18 A 5 μm 250/4 column) inFIG. 3.

Twice the volume of conjugation buffer was added to the ligation buffer(0.1 M Na₂HPO₄, 20 mM TCEP, 50 mM MesNa, pH=7). Modified sepharose beadswere subsequently added and the two-phase system was shaken for 30minutes. The supernatant of the sepharose gel was analysed using UPLC-MS(FIG. 3, chromatogram 2); no mass that could be assigned to the ligationproduct could be found in the connected mass spectrometer. However,since an absorption signal can be observed at the retention time of theproduct, it also becomes apparent that impurities can be removed thatcould not be separated by means of HPLC. After washing with water andsome ethanol and acetonitrile, 0.5 vol. % hydrazine hydrate in water wasadded to the peptide-loaded sepharose beads and the supernatant wasanalysed after 30 minutes, wherein the chromatogram 3 (FIG. 3) wasobtained. This shows a high peptide purity (desired product) of over90%.

Example 3: Exemplary Purification of a Peptide Mixture after Solid PhasePeptide Synthesis

The reaction scheme (scheme 5) of purification according to theinvention is shown in FIG. 8.

After solid phase synthesis (SPPS), a capture molecule 2 (FIG. 8: scheme5) is added to the last coupled amino acid. An important prerequisitefor this is that acetylation was complete in the previous steps.Afterwards, all truncated sequences and the capture-molecule-modifiedfull-length peptide are cleaved from the resin. Subsequently,non-peptide impurities are removed by ether precipitation and the crudepeptide mixture is dissolved in an acetate buffer which has a pH valueof 3-4 and to which 0.1 M aniline is added as catalyst. This solution isnow added to functionalized sepharose beads 1 (FIGS. 8 and 9). Thismaterial is applied in protein purification as a material for affinitychromatography. Sepharose has the advantage of being easily penetratableby the peptides. The sepharose is previously aldehyde modified (see FIG.9), the aldehyde modification of the sepharose is known from: J. Guisan,Enzyme Microb. Technol. 1988, 10, p. 375.

Hereby, only peptides that carry the capture molecule with the hydrazidefunction are anchored to the solid support. Amines, which theoreticallycan also react with aldehydes, are protonated at the pH value to be usedand therefore not nucleophilic enough for an attack on aldehyde. Thetruncated sequences that are still in the sepharose can be washed outwith water. Treatment of sepharose with a basic solution, e.g. ammoniain water, causes the capture molecule to decompose and the full-lengthpeptide dissolves. The solution can then be lyophilized, wherein ammoniais removed. Subsequently, the peptide is obtained in pure form as asolid. One advantage of the method is its rapid immobilization and broadapplicability to different peptides.

Example 4: Exemplary Regeneration of the Purification Resin

After peptide purification with capture molecule 2 (FIG. 8: scheme 5),the original aldehyde function of 1 remains blocked with hydrazine, inorder to make the purification resin available again for a newpurification cycle, the resin needs to be regenerated and thus thealdehyde function needs to be restored. This is achieved by shifting theequilibrium by adding aldehydes or ketones. The exemplary feasibilitywas shown as follows. The purification resin 1 was divided into threeequal aliquots (I, II, III) and two aliquots (II, III) were treated withhydrazine in conjugation buffer for 30 min. Subsequently, it was washedwith water and Aliquot III was washed seven times with a mixture ofwater, acetone and TFA (49.5:49.5:1). Then all aliquots were treatedwith FmocN₂H₃ in conjugation buffer for 14 h and then each washed fivetimes with water, DMF and water. This was followed by two treatmentswith DMF/Piperidine (20%) for 4 min each and then the UV absorption ofthe resulting fluoren piperidine adduct was measured at 301 nm in thesupernatant (SmartSpec™Plus Spectrophotometer from BioRad in 1 mLsemi-micro quartz cuvettes from Hellma). The untreated aliquot I showeda load of 27 μmol/g; the aliquot II without regeneration a load of 18μmol/g (67% of I), and the regenerated resin showed a load density of 25μmol/g (93% of I). This experiment shows the successful regeneration ofthe purification resin.

TABLE 1 UV-absorption and loading density of purification resins afterdifferent regeneration treatments A S Proportion Aliquot (301 nm)(μmol/g) [%] I 0.286 27 100 II 0.199 18 67 III 0.267 25 93

Example 5: Purification of a Peptide Mixture

The method according to the invention for the purification of peptideswas applied to seven peptides of different polarity, which wereH-TLADEVSASLAK-OH (SEQ ID NO: 3) (7) fragment 427-438 of the tau proteinrelevant to Alzheimer's disease, H-ATLADEVSASLAK-NH₂ (SEQ ID NO: 4) (8)fragment 427-439 of tau, the cysteinyl peptideH-CQWSLHRKRHLARTLLTAAREPRPAPPSSNKV-NH₂ (SEQ ID NO: 5) (8) from proteinprogona-doliberin-2 (9), H-GIGKFLHSAKKFGKAFVGEIMNS-NH₂ (SEQ ID NO: 6)Magainin (10), H-YLFFYRKSV-NH₂ Terts72Y (SEQ ID NO: 7) (11),H-FPRPGGGGNGDFEEIPEEYL-NH₂ (SEQ ID NO: 8) Bivalirudin (12) andH-GRKKRRQRRRPQ-NH₂ TAT (SEQ ID NO: 9) (13).

The crude peptide mixture was dissolved in the conjugation buffer andadded to the sepharose beads 1 within 30-60 min, subsequent washing withwater and neutral aqueous solutions (4M urea, 1M table salt) allacetylated truncated sequences and other impurities could be removed.The cleavage of linker 2 (FIG. 10: scheme 7) was performed with 5%ammonia solution in water for 20 minutes, it was then neutralizedin-situ with acetic acid and the solubility was increased.

The purity of the individual phases was assessed by means of theUPLC-MS. The chromatograms of the non-purified (without capture moleculeFM) and purified peptides as well as of the supernatant that containedthe impurities are shown in FIG. 4. A peptide purity of 85% (originally39%) was achieved for 7, of 93% (originally 39%) was achieved for 8, of80% (originally 24%) was achieved for 9, of 90% (originally 23%) wasachieved for 10, of 87% (originally 60%) was achieved for 11, of 90%(originally 40%) was achieved for 12, of 95% (originally 37%) wasachieved for 13 (see FIG. 4).

TABLE 2 Purity and yield of different peptides after application of thepurification meethod according to the invention Hydro Purity PurityLength phobic crude after Name No. Application Status in aa aa producttreatment Yield Tau1 7 Alzheimer's preclinical 13 62% 40% 85% 70%disease Tau2 8 Alzheimer's preclinical 12 58% 39% 93% 69% disease GNRH 9fertilisation approved 32 59% 35% 80% 42% Magainine 10 antibioticapproved 23 65% 23% 90% 62% Terts72Y 11 lung cancer phase II 9 77% 60%87% 92% Bivalrudin 12 anti- approved 20 65% 40% 90% 89% coagulation TAT13 HIV phase II 12 17% 37% 95% 83% Testpeptid 14 research — 13 46% 45%60% —

The synthesis of the base-labile linker 2 was performed according toscheme 7 in FIG. 10.

Example 6: Modification of Peptides

General Methods

Solid Phase Peptide Synthesis (SPPS) Linker Coupling and TFA-Cleavage:

The peptide sequences P1-P3 (H-CRVPGDAHHADSLC-NH₂ (SEQ ID NO: 11) (P1),H-VRCPGAAHHADSLC-NH₂ (SEQ ID NO: 12) (P2), H-VRVPGCAHCADSLY-NH₂ (SEQ IDNO: 13) (P3)) were synthesized in 100 μmol scale under standard solidphase peptide synthesis conditions on a Intavis MultiPep RSi, wherebythe synthetic resin was treated with acetic anhydride and pyridine aftereach amino-acid coupling to block unreacted amino groups. The resin waspreswollen for 15 min prior to linker coupling. Linker (14) was coupledto P1-P3 on resin by usage of 4.75 eq linker (14) (281 mg), 8.75 eqoxyma (124 mg) and 8.5 eq diisopropylamine (DIEA, 147 μL) for 2 h in 1.3mL DMF. The amounts used were based on the loading of the SPPS resin(Rinkamide RAM). The reaction mixture was shaken for 120 min, filteredoff, washed (3×DMF, 3×DCM) and dried.

The dried resin was incubated with TFA-Cleavage cocktail (Reagent K:TFA/H₂O/PhOH/PhSH/Ethandithiol, 82.5:5:5:52.5, v/v/w/v/v, 10 mL per 100μmol synthetic scale) for 120 min. The cleavage solution was separatedfrom the synthesis resin by filtration and collected in a Falcon tube 10times the volume of the solution. By adding 9-fold the volume ofrefrigerated diethyl ether, the peptide was precipitated. The vessel wascentrifuged at 5000 rpm for 3.5 min, the supernatant solution decantedoff and the peptide pellet washed again with diethyl ether followed bydecantation. The peptides were then dissolved in H₂O/MeCN/TFA,(99.95:99.95:0.1, v/v/v), frozen with liquid nitrogen and lyophilizedunder high vacuum.

General Procedure to Purify and/or Cyclize Peptides by the InventiveMethod

Dissolution and Immobilization of Crude Linker-Tagged Peptides

The crude linker-tagged peptide mixture was dissolved in pure DMSO (4.5mL for 100 μmol synthetic scale). After complete dissolution 10 vol. %Immobilisation Buffer 2 (0.1 M citric Acid/Na₂CO₃ pH 3.5 with 8 Mguanidinium chloride, 0.45 mL for 100 μmol synthetic scale) was added.

The linker-tagged peptide is immobilized on 1.5 times the amount ofaldehyde-modified agarose loaded with 100 μmol/1 mL settled beads (100μmol/2 mL of slurried beads) or on hydroxylated poly(methyl acrylate)beads. After aliquoting, the beads material is washed with 3× milli-Qwater and 3× immobilization buffer 1 (0.1 M citric Acid/Na₂CO₃ pH 4.5).The dissolved crude linker-tagged peptide mixture was then added to theagarose and shaken. Of note, the beads should have a free and goodfluctuation in the immobilization solution. After a reaction time of 90minutes, the immobilization solution is filtered off with suction.

Washing

After immobilization, the supernatant was removed and beadslinker-connected to the peptides were washed each, three-times 5 mL (for100 μmol scale) with DMSO. Thereafter a mixture of L-Cysteine in 0.1 Mcitric Acid/Na₂CO₃ pH 4.5 is added, and reactors are shaken for 15 minor longer. This mixture was removed, and beads were washed three-timeswith 1 mL (for 10 μmol scale) with the following solvents and solutions:

-   -   1) DMSO    -   2) 6 M guanidinium in water    -   3) 70% ethanol in with 0.1 M NaCl Milli-Q water    -   4) Water    -   5) MeCN

Purification or Cyclization

If peptides were just to be purified no cyclization step was performed.

In case of cyclization by disulfide formation, after immobilization andwashing of target peptide, disulfide were formed in a 1 to 1 mixture ofDMSO and 0.4 M (NH₄)₂CO₃, 6M GdmCl on the support. Thereafter theoxidation buffer was washed out and the peptide is released as describedbelow.

Release of Peptides

To the beads 200 μl of 0.2 M ethanolamine or methylamine cleavagesolution (pH 11) was added, incubated for 5 min, and collected in acentrifuge tube. Further 600 μL 0.2 M ethanolamine cleavage solution (pH11) was added to the beads and shaken for 60 min. Thereafter thesolution was collected in the same centrifuge tube. Peptides werefurther eluted with 2×500 μL H₂O/MeCN (7:3; 0.1% AcOH) and with 2×500 μLH₂O/MeCN (3:7; 0.1% AcOH) to the same tube. The peptide sample werefrozen with liquid nitrogen and lyophilize to gain a white amorphouspeptide powder.

Example 6a: Formation of Macrocycle of Peptide P1-P3 Bound toPurification Solid Support by Disulfide (Intramolecular Cyclization)

The inventive method for the immobilization, modification andpurification of peptides was applied to the peptide H-CRVPGDAHHADSLC-NH₂(SEQ ID NO: 11) (P1), H-VRCPGAAHHADSLC-NH₂ (SEQ ID NO: 12) (P2),H-VRVPGCAHCADSLY-NH₂ (SEQ ID NO: 13) (P3), (underlined C residues areconnected as a disulfide): The three peptides were synthesized asdescribed above. Before linker coupling an aliquot of resin (1 mg) wastaken and peptide has been released by TFA (Reagent K, 2 h). Thus, thecrude chromatogram before purification has been recorded after Et₂Oprecipitation and Et₂O wash (see crude purities in table 1). Thereafterlinker (14) was coupled as described above. The linker tagged crudepeptide mixture was gained by TFA treatment as described above at thegeneral method (Reagent K, 2 h). After precipitation and washing withEt2O, the precipitate was dried.

The aldehyde functionalized agarose beads were filled in cartridges andthe material was washed with 3× milli-Q water and 3× immobilizationbuffer 1 (0.1 M citric Acid/Na₂CO₃ pH 4.5). 113 μl of DMSO was added tothe beads and soaked for 5 min. Each 6 mg of the crude linker-taggedpeptides (P1-3) was dissolved in 223 μl of DMSO and 26 μl Buffer 2 (0.1M citric Acid/Na₂CO₃, 7 M guanidin-hydrochlorid pH 3.5) and the mixturewas then added to the crosslinked agarose beads and shaken. Of note, thebeads should have a free and good fluctuation in the immobilizationsolution. After a reaction time of 2 h, the immobilization solution isfiltered off with suction.

Washing: After immobilization, the beads linker-connected to thepeptides were washed each, three-times 5 mL (for 2.5 μmol scale) withthe following solvents and solutions

-   -   1) DMSO    -   2) 6 M guanidinium in water    -   3) 70% ethanol in with 0.1 M NaCl Milli-Q water    -   4) Water    -   5) MeCN

For oxidation of cysteine residues 500 μL of DMSO was added to the beadsand the cartridges were shaken for a total of 24 h. Beads were washed 3×water and twice with MeCN.

To the beads 200 μl of 0.2 M ethanolamine cleavage solution (pH 11) wasadded, incubated for 5 min, and collected in a centrifuge tube. Further600 μL 0.2 M ethanolamine cleavage solution (pH 11) was added to thebeads and shaken for 60 min. Thereafter the solution was collected inthe same centrifuge tube. Peptides were further eluted with 2×500 μLH₂O/MeCN (7:3; 0.1% AcOH) and with 2×500 μL H₂O/MeCN (3:7; 0.1% AcOH) tothe same tube. From this tube analytical samples were taken diluted andanalyzed. The gained amounts and masses can be found in table 1.

TABLE 1 crude UV₂₁₀ purity/ purity after modification yield of sequence*purity of cycliza- No. after modification linker-peptide tioncalculated vs. found ESI masses P1 S-----------------------S 72%/ <99%MH²⁺calc.: 739.83 m/z, MH³⁺calc.: 493.56 m/z H-CRVPGDAHHADSLC- 67%MH²⁺found.: 739.81 m/z, MH³⁺found.: 493.48 m/z NH₂ P2S-------------------S 69%/ <99%MH²⁺calc.: 717.33 m/z, MH³⁺calc.: 478.56 m/z H-VRCPGAAHHADSLC- 53%MH²⁺found.: 717.80 m/z, MH³⁺found.: 478.87 m/z NH₂ P3 S---S 59%/ <99%MH²⁺calc.: 744.35 m/z, MH³⁺calc.: 496.57 m/z H-VRVPGCAHCADSLY- 56%MH²⁺found.: 744.83 m/z, MH³⁺found.: 496.76 m/z NH₂

Example 6b: Purification of P4 by Inventive Method

The inventive method for the immobilization and purification of peptideswas applied to the peptide from HIV reverse transcriptase (159-173)H-ELRQHLLRWGLTTPD-NH₂ (SEQ ID NO: 14) (P4). The fragment [159-173] ofHIV reverse transcriptase has been synthesized automatically onRink-Amide polystyrene resin in 10 μmol scale on MultiPep RSIsynthesizer. An aliquot of resin (1 mg) was taken, and peptide has beenreleased by TFA (Reagent K, 2 h). Thus, the crude chromatogram beforepurification has been recorded after Et₂O precipitation and Et₂O wash,showing a crude purity of 34% (abs. 210 nm). Thereafter base-labilelinker (14) was coupled on the rest of resin as described above underGeneral Methods. Thereafter the linker-tagged peptide has been detachedby TFA (Reagent K, 2 h), yielding 20.3 mg crude peptide after Et₂Oprecipitation, a single Et₂O wash and drying under reduced pressure(high vacuum).

The crude peptide was dissolved by addition of 500 μL MeCN andsubsequent addition of 500 μL buffer 1 (0.1 M citric Acid/Na₂CO₃ pH4.5). Purification has been performed with methylamine as describedabove under General Methods/Release of Peptides, yielding 6.2 mg (2.9μmol, 98% purity) in a yield of 29% and a recovery of 85%.

UPLC: t_(R)=1.92 min (gradient: 0-90% B, 3 min).

ESI-MS: 917.76 m/z, calc. [M+H]²⁺, 917.51, C₈₂H₁₃₂N₂₆O₂₅, calculated MW:1834.12 g·mol⁻¹.

Materials and Methods

Solid Phase Peptide Synthesis and Purification

The automated solid phase peptide synthesis was performed in 25 μmolbatches with a MultiPep RS peptide synthesis machine from Intavis AG.The syntheses of peptide amides were performed on Tentagel R RAM resin(0.2 mmol-g-1) from Rapp Polymer. Before the beginning of the synthesis,the resin was transferred to 3 mL syringe reactors (PE reactor fromMultisyntech) and soaked in DMF. Unless otherwise specified, thespecification of equivalents refers to the initial loading of the resinused.

Fmoc-Removal:

To remove the temporary Fmoc protecting groups, the resin was treatedonce for 4 min and once for 6 min with 400 μL piperidine/DMF (4:1) andsubsequently washed five times with 800 μL DMF and it was continued withthe coupling of the Fmoc amino acid derivatives.

Coupling of Fmoc Amino Acid Derivatives:

A solution of 5 eq. amino acid in DMF (0.3 M) was pre-activated for 1min at room temperature with solutions of 4.5 eq. HCTU in DMF (0.3 M)and 10 eq. NMM in DMF (0.6M) and then added to the resin. After 30 minreaction time, the resin was washed three times with 800 μL DMF and itwas continued with blocking the termination sequences.

Blocking the Truncated Sequences:

The resin was treated once for 5 min with 400 μl AC₂O/2,6-Lutidin/DMFsolution (5:6:89) and subsequently washed three times with 800 μL DMFeach.

Last Step Coupling of the Capture Molecule:

As the last step of the solid phase peptide synthesis, the capturemolecule 2 was coupled to the desired target peptide. The synthesisresin was mixed with a solution of 5 eq. capture molecule 2 in DMF (0.3M) and 5 eq. Oxyma in DMF (0.3 M) and 12 eq. DIPEA in DMF (0.7 M) mixed.After 60 min reaction time, it was washed twice with 800 μL DMF each.

Release from Polymeric Support:

The resin was with 2 mL of a solution of 96% TFA, 2% water, 2%triisopropylsilane, in the case of thiol-containing amino acids(cysteine or methionine) in the target sequence 0.5% ethanedithiol and0.5% thioanisole were added to the solution, wherein the amount of TFAwas reduced by 1%. The synthesis resin was treated with this cleavingmixture and shaken for 2 h at room temperature. Afterwards, the cleavingsolution was collected and the resin was washed twice with 1 mL TFAeach. The cleaving solution was combined with the washing solutions andprecipitated in 50 ml cold diethyl ether. This suspension was thencentrifuged and the organic supernatant was discarded.

Immobilization on Purification Resin:

After centrifugation, the crude precipitate was dissolved in 3 ml of theconjugation buffer (0.1 M NH₄OAc, 0.1 M aniline, pH=3), if the mixturedid not dissolve completely, acetonitrile was added. This solution wastransferred into a 6 ml syringe from bBraun with a filter insert PE 25μm pore size from Multisyntech, in this syringe was one gramfunctionalized sepharose. Immobilization was performed for 30 to 60minutes. It was then washed 5 times with deionized water of MilliQpurity, 5 times with a 4M urea solution and 5 times with water. Then thedesired peptide was cleaved basically with 5 v % NH₄OH and 1 v %mercaptoethanol in water from the resin. Lyophilization provided thedesired peptides as a white flaky solid.

Synthesis of Capture Molecule 2 (Compound 2 in Scheme 7—Corresponds toCompound (25)):

tert-butylhydrazine Carboxylate 5 (Compound 5 in Scheme 7)

Hydrazine monohydrate (80%, 32.5 g, 520 mmol) was mixed with isopropanol(100 ml) at 0° C., with a solution of Boc₂O (50.0 g, 230 mmol) inisopropanol (50 ml) drop by drop. The reaction mixture became turbidafter addition and stirring was continued at room temperature for 2 h.The solvent was removed, the residue dissolved in dichloromethane anddried over magnesium sulphate. Then the solvent was evaporated and theresidue was recrystallized from hexane, resulting in the title compound5 (22.8 g, 75%) as colorless crystals. Smp 36-37° C. Rf (EtOAc/hexane1:1) 0.20. ¹H-NMR (300 MHz, CDCl₃): δ 6.16 (s, 1H, NH), 3.67 (s, 2H,NH₂), 1.42 (s, 9H, C(CH₃)₃). ¹³C-NMR (75 MHz, CDCl₃, TMS): δ 158.3,77.2.28.5. The analytical data are consistent with the literature data(A. Bredihhin, U. Mäeorg, Tetrahedron 2008, 64, 6788-6793).

Bis(4-nitrophenyl)(thiobis(ethane-2,1-diyl))bis(carbonate) 6 (Compound 6in Scheme 7)

6.72 (33 mmol) 4-nitrophenyl chloroformate was added to a solution of1.87 ml (2.12 g, 15 mmol) 2,2-thiodiethanol in 40 ml anhydrousdichloromethane. Then 2.68 ml (33 mmol) of anhydrous pyridine was slowlyadded drop by drop with ice cooling and vigorous stirring. The reactionmixture was stirred for 1 h at room temperature. The reaction solutionwas mixed with 100 ml saturated ammonium chloride solution, extractedthree times with 100 ml chloroform and dried over anhydrous magnesiumsulphate. The organic phases were combined and constricted in a vacuum.The residue was absorbed into ethyl acetate and the product wasprecipitated with a small amount of cyclohexane. After filtration, 5.43g (12 mmol, 80%) was obtained as white solid. Melting point: 136.5° C.,Rf (EtOAc/cyclohexane 1:1) 0.78. ¹H NMR (300 MHz, DMSO) δ 8.30 (d, J=9.2Hz, 2H, Ar—H), 7.55 (d, J=9.3 Hz, 2H, Ar—H), 4.42 (t, J=6.4 Hz, 2H,CH₂), 2.96 (t, J=6.5 Hz, 2H, CH₂). ¹³C-NMR (75 MHz, CDCl₃, TMS): δ155.22, 151.93, 145.17, 125.41, 122.56, 67.85, 29.63.

2-[2-(1-((tert-butyl)oxy-carbonyl)oxy-carbonyl)-hydrazyl-ethylsulfanyl]-ethyl4-nitrophenyl Carbonate 7 (Compound 7 in Scheme 7)

1.97 g (4.31 mmol) bis(4-nitrophenyl)(thiobis(ethane-2,1-diyl))bis(carbonate) 6 were added to 20 ml drydichloromethane and at 0° C. 1 eq. (0.58 g, 4.31 mmol) tert-butylhydrazine carboxylate 5 with 3 eq. (1.13 ml, 6.66 mmol) DIPEA was slowlyadded dropwise for one hour. The reaction solution was stirred foranother 12 hours and then mixed with water. The product was extractedthree times with 100 ml dichloromethane and dried over anhydrousmagnesium sulphate. The organic phases were combined and constricted ina vacuum. The residue was purified by column chromatography(EtOAc/cyclohexane 2:1), after which 1.03 g (2.31 mmol, 53%) of atransparent oil was obtained. Rf (EtOAc/Cyc10hexane 1:1) 0.20. ¹H NMR(300 MHz, CDCh) δ 8.28 (d, J=9.1 Hz, 2H, Ar—H), 7.39 (d, J=9.1 Hz, 1H,Ar—H), 6.64 (s, 1H, NH), 6.33 (s, 1H, NH), 4.44 (t, J=6.8 Hz, 2H, CH₂),4.33 (t, J=6.6 Hz, 2H, CH₂), 2.92 (t, J=6.8 Hz, 2H, CH₂), 2.84 (t, J=6.6Hz, 2H, CH₂), 1.46 (s, 9H). ¹³C NMR (75 MHz, CDCl₃) δ 155.52, 152.57,125.47, 121.96, 82.09, 68.06, 65.34, 64.18, 31.10, 30.59, 28.27, 27.03.

2-[2-(1-((tert-butyl)oxy-carbonyl)oxy-carbonyl)-hydrazyl-ethylsulfonyl]-ethyl4-nitrophenylCarbonate 2 (Compound 2 in Scheme 7)

To a solution of thioether 7 (0.52 g, 1.1 mmol) in 50 mldichloromethane, 77% (489 g, 2.2 mmol) of m-CPBA was slowly added atroom temperature. After stirring for 12 h the reaction mixture was mixedwith 1M NaHCO₃ solution and the organic phase was extracted three timeswith 50 ml dichloromethane. The combined organic phases were dried withmagnesium sulphate and the solvent was removed from the rotaryevaporator, after which the product was precipitated as white amorphoussolid 0.52 mg (1.1 mmol, quantitative). ¹H NMR (300 MHz, CDCl₃) δ 8.29(d, J=9.2 Hz, 2H, Ar—H), 7.41 (d, J=9.2 Hz, 2H, Ar—H), 6.89 (s, 1H, NH),6.36 (s, 1H, NH), 4.74 (t, J=5.9 Hz, 2H, CH₂), 4.63 (t, J=5.3 Hz, 2H,CH₂), 3.54 (t, J=5.9 Hz, 2H, CH₂), 3.45 (t, J=5.5 Hz, 2H, CH₂), 1.45 (s,9H). ¹³C NMR (75 MHz, CDCl3) δ 155.85, 155.18, 152.29, 145.79, 125.56,122.00, 82.32, 62.25, 59.45, 54.01, 53.24, 28.23.

The synthesis of the compounds according to formula (14), (15), (17),(18), (19): was based on a modular principle, which is shown in thegeneral synthesis scheme (Scheme 1).

Syntheses for the Preparation of the Compound of Formula (14)

N,N′-Bis-(tert-butoxycarbonyl)-aminooxyacetyl-N-hydroxylsuccinimideEster (BS2: X=O, R1=Boc)

Add N-hydroxylsuccinimide (0.41 g, 3.20 mmol) anddicyclohexylcarbodiimide (0.67 g, 0.32 mmol) at 0° C. to a solution ofcommercially available N,N′-bis-boc-amino-oxyacetic acid (1.00 g, 3.20mmol) in 11 ml ethyl acetate/dioxane (1:1). At room temperature thesolution was allowed to stir for 3 hours and the suspension was filteredover Celite and washed with ethyl acetate. The filtrate was concentratedunder vacuum to dry and dissolved again in 100 ml ethyl acetate. It waswashed with 5% NaHCO₃ solution, saturated NaCl solution and water (100ml each). The organic phase was dried with MgSO₄ and evaporated undervacuum wherein 1.24 g (3.20 mmol) product was obtained as a white solid.Yield: 1.24 g (quant.); Rf (cyclohexane/ethyl acetate, 1:1) 0.50; ¹H NMR(300 MHz, CDCl₃) δ 4.86 (s, 2H), 2.85 (s, 4H), 1.53 (s, 18H).

N,N′-Bis-(tert-butoxycarbonyl)-aminooxyacetyl-1-((2-aminoethyl)thio)propan-2-olamide (BS3: X=O, R1=Boc)

BS2 (X=O, R₁=Boc, 1.00 g, 2.55 mmol) was mixed in 25 ml dichloromethanewith 1-((2-aminoethyl)thio)propan-2-ol (0.38 g, 2.55 mmol) anddiisopropyl-ethylamine (DIPEA, 0.53 ml, 3.06 mmol) and stirredovernight. It was washed with 5% NaHCO₃ solution, saturated NaClsolution and water (100 ml each). The organic phase was dried with MgSO₄and vacuum-constricted wherein 1.04 g (2.55 mmol) product was obtainedas a white solid. Yield: 1.04 g (quant.); Rf (CH₂Cl₂/MeOH, 98:2) 0.35;¹H NMR (300 MHz, CDCl₃) δ 7.95 (s, 1H), 4.44 (s, 2H), 3.92-3.82 (m, 1H),3.53 (qd, J=6.7, 1.5 Hz, 2H), 2.83-2.65 (m, 4H), 2.49 (dd, J=13.7, 8.7Hz, 1H), 1.55 (s, 18H), 1.25 (d, J=6.2 Hz, 3H); ¹³C NMR (75 MHz, CDCl₃)δ 167.94, 150.57, 85.41, 65.98, 41.86, 38.81, 32.21, 28.19, 22.23.

2,2-Dimethylpropanoyloxy-[2-[2-[2-[2-(4-nitrophenoxy)carbonyloxypropylthionyl]ethylamino]-2-oxo-ethoxy]amino]2,2-dimethylpropanoate P′ (X=O, R₁=Boc, R₂=OC₆H₄pNO₂)

Bis(4-nitrophenyl)carbonate (1.01 g, 4.95 mmol) were added to a solutionof BS3 (X=O, R₁=Boc) (1.52 g, 3.30 mmol) in 5 ml dry CH₂Cl₂. Drypyridine (0.40 ml, 4.95 mmol) was then added under ice cooling. Thereaction solution was stirred for 18 hours. The precipitate was filteredoff and was washed with 50 ml DCM. The filtrate was washed withsaturated NH₄Cl solution (50 ml) and the aqueous phase was extractedwith 50 ml CHCl₃. After drying with MgSO₄, the combined organic phaseswere constricted on the rotary evaporator and the residue was purifiedby column chromatography (cyclohexane/etOAc, 2:1). Yield: 1.27 g (67%);Rf (cyclohexane/etOAc, 1:1) 0.44; ¹H NMR (300 MHz, CDCl₃) δ 8.27 (d,J=9.2 Hz, 2H), 7.89 (s, 1H), 7.39 (d, J=9.2 Hz, 2H), 5.00 (dd, J=12.5,6.3 Hz, 1H), 4.43 (s, 2H), 3.53 (dd, J=13.3, 6.5 Hz, 1H), 2.86-2.69 (m,2H), 1.53 (s, 18H), 1.47 (d, J=6.3 Hz, 3H).

2,2-Dimethylpropanoyloxy-[2-[2-[2-(4-nitrophenoxy)carbonyloxypropylsulfonyl]ethylamino]-2-oxo-ethoxy]amino]2,2-dimethylpropanoates Formula (14)

Slowly add m-CPBA (0.96 g, 4.30 mmol) at room temperature to a solutionof P′ (X=O, R₁=Boc, R₂=OC₆H₄pNO₂) (1.27 g, 2.15 mmol) in 21 mldichloromethane. After 12 hours of stirring, the reaction mixture waswashed twice with a saturated NaHCO₃ solution (15 ml) and the organicphase was concentrated in a vacuum after drying with MgSO₄. The sulfonewas preserved as a white solid. Yield: 1.26 g (97%); Rf(cyclohexane/etOAc, 1:1) 0.29; ¹H NMR (300 MHz, CDCl₃) δ 8.28 (d, J=9.3Hz, 1H), 8.15 (t, J=5.8 Hz, 1H), 7.41 (d, J=9.3 Hz, 1H), 5.48-5.39 (m,1H), 4.44 (s, 1H), 3.83 (dd, J=6.2, 4.2 Hz, 1H), 3.56 (dd, J=14.9, 8.3Hz, 1H), 3.37 (t, J=6.5 Hz, 1H), 3.27 (dd, J=14.9, 3.9 Hz, 1H), 1.57 (d,J=6.4 Hz, 1H), 1.54 (s, 7H). ¹³C NMR (75 MHz, CDCl₃) δ 168.58, 155.42,151.58, 150.52, 145.67, 125.46, 122.04, 85.60, 77.16, 76.62, 70.63,58.19, 53.67, 33.07, 28.16, 20.21; ESI-MS: (calculated MNa⁺: 628.16g/mol, found: 628.17 m/z).

Syntheses for the Preparation of the Compound of Formula (18)

((tert-butoxycarbonyl)amino)glycine (BS1 X=NH, R₁=H)

Bromoacetic acid (1.48 g, 10.4 mmol) was added to a methanolic solution(10 ml) of NaOH (0.70 g, 17.4 mmol) and Boc-hydrazine (1.17 g, 8.7 mmol)at 0° C. The solution was heated for 5 hours under reflux. Then MeOH wasremoved and 50 ml water was added. The aqueous phase was extracted threetimes with ethyl acetate (50 ml). The aqueous phase was then brought topH 2 with citric acid and extracted three times with 50 ml ethylacetate. The combined organic phases were dried with MgSO₄ and thesolvent was removed under reduced pressure. Yield: 0.85 g (51%) whitesolid; Rf (CH₂Cl₂/MeOH, 8:2) 0.15. ¹H NMR (300 MHz, DMSO) δ 8.55 (s,2H), 8.17 (s, 2H), 3.40 (s, 2H), 1.37 (s, 9H); ESI-MS: (calculated MH⁺:191.10 g/mol, found: 191.33 m/z).

N-(tert-butoxycarbonyl)-N-((tert-butoxycarbonyl)amino)glycine (BS1X=NBoc, R₁=H)

Boc₂O (5.74 g, 26.03 mmol) was added as a solid to a solution of((tert-butoxycarbonyl)amino)glycine (5.00 g, 26.0 mmol) and NaOH (1.57g, 39.04 mmol) in 104 ml dioxane/H₂O (1:1). The solution was stirredovernight at room temperature for 18 hours and the dioxane was thenremoved under reduced pressure. Add 100 ml saturated NaHCO₃ solution tothe aqueous residue and wash twice with 100 ml Et₂O. The aqueous phasewas brought to pH 2 with citric acid. The white suspension was extractedthree times with 150 ml ethyl acetate. After drying with MgSO4 thesolvent was removed wherein a white solid formed. Yield: 7.56 g(quant.); Rf (CH₂Cl₂/MeOH, 9:1) 0.75; ¹H NMR (300 MHz, DMSO) δ 12.34 (s,1H), 9.24 (s, 1H), 3.56 (s, 2H), 1.46-1.32 (m, 18H).

N-(tert-butoxycarbonyl)-N-((tert-butoxycarbonyl)amino)glycinyl-N-hydroxylsuccinimide(BS2 X=NBoc, R₁=H)

N-(tert-butoxycarbonyl)-N-((tert-butoxycarbonyl)amino)glycine (1.36 g,4.45 mmol) in 15 ml ethyl acetate/dioxane (1:1) was added at 0° C. toN-hydroxylsuccinimide (0.52 g, 4.45 mmol) and dicyclohexylcarbodiimide(DCC, 0.93 g, 4.45 mmol). At room temperature, the solution was allowedto stir for 15 hours. Afterwards the suspension was filtered over Celiteand washed with ethyl acetate. The filtrate was concentrated undervacuum to dry and dissolved again in 100 ml ethyl acetate. It was washedwith 5% NaHCO₃ solution, saturated NaCl solution and water (100 mleach). The organic phase was dried with MgSO₄ and evaporated undervacuum wherein 1.24 g (3.20 mmol) product was obtained as white foam.Yield: 1.51 g (88%); Rf (cyclohexane/ethyl acetate, 1:1) 0.45; ¹H NMR(300 MHz, CDCl₃) δ 4.67 (s, 1H), 4.19 (s, 2H), 2.87 (s, 4H), 1.49 (m,18H).

N-(tert-butoxycarbonyl)-N-((tert-butoxycarbonyl)amino)glycinyl-1-((2-aminoethyl)thio)propan-2-olAmide (BS3: X=NBoc, R₁=H)

BS2 (X=NBoc, R₁=H, 0.36 g, 0.92 mmol) was mixed in 10 ml dichloromethanewith 1-((2-aminoethyl)thio)propan-2-ol (0.13 g, 0.92 mmol) and DIPEA(0.18 ml, 1.01 mmol) and stirred overnight. It was washed with 5% NaHCO₃solution, saturated NaCl solution and water (100 ml each). The organicphase was dried with MgSO₄ and vacuum-constricted wherein 0.27 g (0.65mmol) product was obtained as white foam. Yield: 0.27 g (quant.); Rf(CH₂Cl₂/MeOH, 98:2) 0.36; ¹H NMR (300 MHz, CDCl3) δ 8.34 (s, 1H), 6.66(s, 1H), 4.05 (s, 2H), 3.90-3.81 (m, 1H), 3.49 (dd, J=10.4, 4.0 Hz, 2H),2.78-2.63 (m, 3H), 2.47 (dd, J=13.7, 8.7 Hz, 1H), 1.49 (s, 9H), 1.46 (s,9H), 1.23 (d, J=6.2 Hz, 3H).

[2-(2,2-dimethylpropanoyloxy)-2-[2-[2-[2-[2-(2,5-dioxopyrrolidin-1-yl)oxycarbonyloxypropylthionyl]ethylamino]-2-oxo-ethyl]hydrazino] 2,2-dimethylpropanoate P′ (X=O,R₁=Boc, R₂=ONO₂C₂H₄)

N,N-disuccinimidyl carbonate (0.19 g, 0.72 mmol) was added to a solutionof BS3 (X=NBoc, R₁=H) (0.25 g, 0.72 mmol) in 5 ml dry CH₂Cl₂. Drypyridine (0.06 ml, 0.73 mmol) was then added under ice cooling. Thereaction solution was stirred for 17 hours. 50 ml DCM was added to thesolution. The organic phase was washed with 10% citric acid solution anddried with MgSO₄. The combined organic phases were constricted at therotary evaporator and the residue was purified by column chromatography(CH₂Cl₂/MeOH, 19:1). Yield: 1.27 g (67%); Rf (CH₂Cl₂/MeOH, 9:1) 0.60; ¹HNMR (300 MHz, CDCl₃) δ 8.53 (d, J=95.5 Hz, 1H), 7.07 (s, J=9.0 Hz, 1H),4.01 (s, 2H), 3.82 (ddd, J=8.2, 6.1, 3.9 Hz, 1H), 3.41 (d, J=6.1 Hz,4H), 2.67 (dt, J=13.2, 9.3 Hz, 4H), 2.46 (dd, J=13.7, 8.2 Hz, 2H), 1.44(s, 9H), 1.41 (s, 9H), 1.18 (t, J=6.8 Hz, 3H). ¹³C NMR (75 MHz, CDCl₃) δ169.71, 162.47, 154.43, 77.16, 66.04, 50.53, 41.57, 39.13, 32.22, 28.18,28.12, 25.57, 22.06, 18.88.

[2-(2,2-dimethylpropanoyloxy)-2-[2-[2-[2-(2,5-dioxopyrrolidin-1-yl)oxycarbonyloxypropylsulfonyl]ethylamino]-2-oxo-ethyl]hydrazino] 2,2-dimethylpropanoate (Formula (18))

m-CPBA (0.81 g, 0.36 mmol) was slowly added at room temperature to asolution of P′ (X=NBoc, R₁=H, R₂=ONO₂C₂H₄) (0.10 g, 0.18 mmol) in 5 mldichloromethane. After stirring for 14 hours, the reaction mixture waswashed three times with 5% NaHCO₃ in a saturated NaCl solution (33 mleach) and the organic phase was then dried with MgSO₄. The solvent wasremoved in a vacuum and the sulfone was preserved as a white amorphoussolid. Yield: 0.08 g (76%); Rf (CH₂Cl₂/MeOH, 9:1) 0.45 1H NMR (300 MHz,CDCl₃) δ 8.48 (s, 1H), 6.95 (s, 1H), 4.39 (dt, J=15.7, 7.8 Hz, 1H), 4.06(s, 2H), 3.73 (d, J=4.3 Hz, 2H), 3.48-3.17 (m, 4H), 3.41 (d, J=6.1 Hz,2H), 3.02 (d, J=13.2 Hz, 1H), 2.85-2.68 (m, 3H), 1.46 (s, 9H), 1.43 (s,9H), 1.30 (d, J=6.4 Hz, 3H); ¹³C NMR (75 MHz, CDCl₃) δ 170.18, 167.99,154.71, 133.30, 131.86, 130.13, 129.83, 128.20, 77.16, 62.88, 53.37,33.53, 28.23, 28.16, 25.57, 23.25. ESI-MS: (calculated MNa⁺: 603.19g/mol, found: 603.06 m/z).

Syntheses for the Preparation of the Compound of Formula (15)

N-(tert-butoxycarbonyl)-aminooxyacetyl-1-((2-aminoethyl)thio)propan-2-olAmide (BS3: X=O, R₁=H)

Commercially available 2-((((tert-butoxycarbonyl)amino)oxy)acetic acid(1.00 g, 4.73 mmol) was dissolved in dry CH₃CN (47 ml). AddN-hydroxysuccinimide (0.66 g, 5.68 mmol) and DCC (1.18 g, 5.68 mmol)successively to the solution and stir the resulting reaction mixture atroom temperature for 1 hour. Then add a solution of1-((2-aminoethyl)thio)propan-2-ol (0.85 g, 5.68 mmol) in 3 ml dry CH₃CNand stir the resulting reaction mixture at room temperature for 18hours. The CH₃CN was removed and the concentrate absorbed in 50 ml ethylacetate. It was washed with 10% citric acid solution (50 ml) andsaturated NaCl solution. The residue obtained was purified bychromatography on a silica gel column with a step gradient of MeOH(1-8%) in CH₂Cl₂ as a mobile phase. The desired building block wasobtained as white foam. Yield: 0.26 g (16%); Rf (CH₂Cl₂/MeOH, 9:1) 0.50;¹H NMR (300 MHz, acetones) δ 8.14 (s, 1H), 4.22 (s, 2H), 3.85 (t, J=1.6Hz, 1H), 3.53-3.38 (m, 2H), 2.72-2.66 (m, 2H), 2.62-2.58 (m, 2H), 1.46(s, 9H), 1.19 (d, J=6.1 Hz, 3H).

[2-[2-[2-(4-nitrophenoxy)carbonyloxypropylthionyl]ethylamino]-2-oxo-ethoxy]amino]2,2-dimethylpropanoate P′ (X=O, R₁=H, R₂=OC₆H₄pNO₂)

Bis(4-nitrophenyl)carbonate (0.276 g, 0.90 mmol) were added to asolution of BS3 (X=O, R₁=H) (0.24 g, 0.75 mmol) in 5 ml dry CH₂Cl₂. Drypyridine (0.07 ml, 0.75 mmol) was then added under ice cooling. Thereaction solution was stirred for 18 hours. The precipitate was filteredoff and was washed with 50 ml DCM. The filtrate was washed withsaturated NH₄Cl solution (50 ml) and the aqueous phase was extractedwith 50 ml CHCl₃. After drying with MgSO₄, the combined organic phaseswere constricted on the rotary evaporator and the residue was purifiedby column chromatography (cyclohexane/etOAc, 2:1). Yield: 0.27 g (96%);Rf (cyclohexane/etOAc, 1:1) 0.34; ¹H NMR (300 MHz, CDCl₃) δ 8.35 (s,1H), 8.27 (d, J=9.1 Hz, 2H), 7.58 (s, 1H), 7.40 (d, J=9.1 Hz, 2H),5.06-4.94 (m, 1H), 4.32 (s, 2H), 3.54 (d, J=6.3 Hz, 2H), 2.87-2.73 (m,4H), 1.48 (d, J=4.1 Hz, 3H), 1.47 (s, Hz, 9H).

[2-[2-[2-(4-nitrophenoxy)carbonyloxypropylsulfonyl]ethylamino]-2-oxo-ethoxy]amino]2,2-dimethylpropanoates Formula (15)

m-CPBA (0.263 g, 1.18 mmol) was slowly added at room temperature to asolution of P′ (X=O, R₁=Boc, R₂=OC₆H₄pNO₂) (0.30 g, 0.59 mmol) in 5 mldichloromethane. After 12 hours of stirring, the reaction mixture waswashed twice with a saturated NaHCO₃ solution (15 ml) and the organicphase was concentrated in a vacuum after drying with MgSO₄. The sulphonewas preserved as white foam. Yield: 0.250 g (84%); Rf(cyclohexane/etOAc, 1:1) 0.05; 1H NMR (300 MHz, CDCl3) δ 8.51 (s, 1H),8.28 (d, J=9.2 Hz, 2H), 7.64 (s, 1H), 7.41 (d, J=9.2 Hz, 3H), 5.47-5.35(m, 1H), 4.33 (s, 2H), 3.88-3.79 (m, 2H), 3.57 (dd, J=14.9, 8.3 Hz, 1H),3.41-3.34 (m, 2H), 3.28 (dd, J=14.8, 3.8 Hz, 1H), 1.57 (d, J=6.4 Hz,3H), 1.48 (s, 9H); ¹³C NMR (75 MHz, CDCl₃) δ 168.58, 155.42, 151.58,150.52, 145.67, 125.46, 122.04, 85.60, 77.16, 76.62, 70.63, 58.19,53.67, 33.07, 28.16, 20.21; ESI-MS: (calculated MH+: 528.16 g/mol,found: 528.15 m/z).

Syntheses for the Preparation of the Compound of Formula (16)

Sodium 4-carboxy-2-nitrobenzenesulfonate

4-sulfamylbenzoic acid was dissolved in a mixture of 5 ml fuming HNO₃and 10 ml H₂SO₄ (95%). The reaction solution was stirred overnight at90° C. and then diluted with 100 ml water. At 0° C. the acid wasneutralized by adding Na₂CO₃. Subsequently, acidification was carriedout by adding HCl until the pH value was 2. The water was removed andthe residue was extracted with EtOH/iPrOH (1:1). Subsequently, theorganic solvent was removed wherein the product was obtained as a brownsolid. Yield: 6.86 g (61%); Rf (CH₂Cl₂/MeOH/AcOH, 7:2:1) 0.05; 1H NMR(300 MHz, MeOD) δ 8.23 (d, J=1.6 Hz, 1H), 8.17 (d, J=1.6 Hz, 1H), 8.16(s, 1H); ESI-MS (neg.): (calculated (M-Na)−: 245.97 g/mol, found: 296.00m/z).

Sodium4-(2-(tert-butoxycarbonyl)hydrazine-1-carbonyl)-2-nitrobenzenesulfonate

A solution of t-butyl carbazate (1.91 g, 14.27 mmol) and sodium4-carboxy-2-nitrobenzenesulfonate (3.84 g, 14.27 mmol) and1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (2.76 g, 14.27 mmol) wasmixed overnight at room temperature in 60 ml methanol/H₂O (1:1). Thesolvents were removed under reduced pressure and the residue waspurified by column chromatography (CH₂Cl₂/MeOH, 9:1). A solution oft-butyl carbazate (1.91 g, 14.27 mmol) and sodium4-carboxy-2-nitrobenzenesulfonate (3.84 g, 14.27 mmol) and1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (2.76 g, 14.27 mmol) wasmixed overnight at room temperature in 60 ml methanol/H₂O (1:1). Thesolvents were removed under reduced pressure and the residue waspurified by column chromatography (CH₂Cl₂/MeOH, 9:1). Yield: 6.86 g(61%); Rf (CH₂Cl₂/MeOH/AcOH, 7:2:1) 0.30; ¹H NMR (300 MHz, DMSO) δ 10.46(s, 1H), 9.04 (s, 1H), 8.01 (d, J=1.5 Hz, 1H), 7.99 (d, J=1.5 Hz, 1H),7.96 (s, 1H), 1.43 (s, 9H); ESI-MS (neg.): (calculated (M-Na)−: 360.05g/mol, found: 360.00 m/z).

tert-butyl 2-(4-(chlorosulfonyl)-3-nitrobenzoyl)hydrazine-1-carboxylate

Cyanuric chloride (0.26 g, 1.42 mmol) was added to a solution of sodium4-(2-(tert-butoxycarbonyl)hydrazine-1-carbonyl)-2-nitrobenzenesulfonate(0.55 g, 1.42 mmol) and 18-crown-6 ether (0.02 g, 0.07 mmol) in dryacetone. The solution was heated for 18 h under reflux. After cooling,the reaction mixture was filtered over Celite and purified by columnchromatography (CH₂Cl₂/MeOH, 9:1). Yield: 0.23 g (43%); ¹H NMR (300 MHz,CDCl₃) δ 10.18 (s, 2H), 8.22 (d, J=0.5 Hz, 1H), 8.22 (dd, J=1.7, 0.5 Hz,1H), 8.18 (d, J=1.6 Hz, 1H), 8.15 (d, J=0.6 Hz, 1H), 1.23 (s, 9H);ESI-MS (neg.): (calculated (M)−: 379.02 g/mol, found: 378.92 m/z).

Syntheses for the Preparation of the Compound of Formula (20)

6-Azidoisobenzofuran-1(3H)-on

A clear solution of 6-amino-phtalid (2.50 g, 15.92 mmol) in 1M HCl (28ml) was cooled to 0° C. and mixed with 3 ml of an aqueous solution ofNaNO₂ (1.66 g, 13.89 mmol) drop by drop. The resulting suspension wasstirred for 10 min at 0° C. and mixed with 10 ml of a solution of NaN₃(2.09 g, 31.85 mmol) drop by drop at 0° C. (strong HN₃ gas development!,foaming). The foamy suspension was stirred at 0° C. for one hour. Theprecipitate was sucked off and washed several times with a total of 300ml of water. The brown solid was crushed and dried overnight in a dryingcabinet. It was then dissolved in 300 ml CH₂Cl₂ and filtered off. Thefiltrate was freed under vacuum from the solvent wherein a light brownsolid was obtained. Yield: 2.53 g (91%); Rf (CH₂Cl₂/MeOH/AcOH, 7:2:1)0.30; ¹H NMR (300 MHz, CDCl₃) δ 7.58 (d, J=1.9 Hz, 1H), 7.47 (dd, J=8.2,0.7 Hz, 1H), 7.31 (dd, J=8.2, 2.1 Hz, 1H), 5.31 (s, 2H); ESI-MS:(calculated (MH)⁺: 176.05 g/mol, found: 176.23 m/z).

5-azido-2-(hydroxymethyl)benzohydrazide

6-Azidoisobenzofuran-1(3H)-on (0.50 g, 2.83 mmol) were dissolved in 6 mldimethylformamide (DMF) and hydrazine hydrate (0.71 ml, 14.13 mmol) wasadded and the solution was stirred at 70° C. for 3 hours. The DMF andhydrazine hydrate were removed under vacuum. The residue was purified bycolumn chromatography (CH₂Cl₂/MeOH, 9:1), wherein a light yellow powderwas obtained. Yield: 0.10 g (17%). Rf (CH₂Cl₂/MeOH, 9:1) 0.53; ¹H NMR(300 MHz, DMSO) δ 9.62 (s, 1H), 7.57 (d, J=8.3 Hz, 1H), 7.20 (dd, J=8.3,2.4 Hz, 1H), 7.09 (d, J=2.4 Hz, 1H), 5.31-5.21 (m, 1H), 4.57 (d, J=5.7Hz, 2H), 4.50 (s, 2H), 3.33 (s, 2H), ESI-MS: (calculated (MH)⁺: 208.08g/mol, found: 207.90 m/z), (calculated (MNa)⁺: 230.07 g/mol, found:230.01 m/z).

tert-butyl 2-(5-azido-2-(hydroxymethyl)benzoyl)hydrazine-1-carboxylate

5-Azido-2-(hydroxymethyl)benzohydrazide (0.10 g, 0.48 mmol) weredissolved in Dioxan/EtOAc/iPrOH (1:1:1) 10 ml and mixed with Bocanhydride (0.105 g, 0.48 mmol) and DIPEA (0.10 ml, 0.57 mmol). Thesolution was stirred at room temperature for 12 hours and then thesolvent was removed under reduced pressure. The residue was absorbed in50 ml CH₂Cl₂ and washed twice with a 10% citric acid solution (50 mleach). After drying with MgSO₄ and removing the organic solvent, ayellowish oil was obtained. Yield: 0.10 g (67%). Rf (CH₂Cl₂/MeOH, 9:1)0.75; ¹H NMR (300 MHz, DMSO) δ 10.06 (s, 1H), 9.04 (d, J=30.3 Hz, 1H),7.69-7.58 (m, 1H), 7.25 (dd, J=8.3, 2.4 Hz, 1H), 7.10 (d, J=1.3 Hz, 1H),5.28 (t, J=5.7 Hz, 1H), 4.62 (d, J=5.6 Hz, 2H), 1.43 (s, 9H); ESI-MS:(calculated (MNa)⁺: 330.12 g/mol, found: 330.29 m/z).

tert-Butyl2-(5-azido-2-(((((2,5-dioxopyrrolidin-1-yl)oxy)carbonyl)oxy)methyl)benzoyl)hydrazin-1-carboxylat

N,N-disuccinimidyl carbonate (0.105 g, 0.41 mmol) was added to asolution of tert-butyl2-(5-azido-2-(hydroxymethyl)benzoyl)hydrazine-1-carboxylate (0.105 g,0.34 mmol) in 5 ml dry dimethylformamide. Then dry pyridine (0.03 ml,0.41 mmol) was added. The reaction solution was stirred for 17 hours atroom temperature. The solvent was removed under vacuum. 50 ml DCM wasadded to the solution. The organic phase was washed with 10% citric acidsolution (2×50 ml) and dried with MgSO₄. The combined organic phaseswere constricted at the rotary evaporator and the residue was purifiedby column chromatography (CH₂Cl₂/MeOH, 19:1). Yield: 0.06 g (40%). Rf(CH₂Cl₂/MeOH, 19:1) 0.45; ¹H NMR (300 MHz, DMSO) δ 10.06 (s, J=10.9 Hz,1H), 8.99 (s, J=8.4 Hz, 1H), 7.62 (d, J=8.3 Hz, 1H), 7.53 (s, 1H), 7.25(dd, J=8.3, 2.4 Hz, 1H), 5.76 (s, 2H), 3.34 (s, 4H), 1.43 (s, 9H),ESI-MS: (calculated (MNa)⁺: 471.12 g/mol, found: 471.25 m/z).

Syntheses for the Preparation of the Compound of Formula (21)

6-Methoxyisobenzofuran-1(3H)-on

A mixture of 3-methoxybenzoic acid (10.00 g, 65.07 mmol), 37% formalinsolution (7.5 ml, 80 mmol), 37% HCl (8.00 ml) and 75 ml 100% acetic acidwas heated for 18 hours with reflux. After cooling, the clear solutionis switched off and left at this temperature for 14 hours. The aceticacid was removed in the air stream at 80° C. The residue was absorbed in150 ml toluene and concentrated to 40 ml. The 80° C. hot solution waswashed with 40 ml portions of 20% Na₂CO₃ solution (3 times) and 40 mlwater.

After adding 3 ml morpholine, the organic phase was stirred for 2 h at80° C. and then washed with 50 ml portions of 10% H₂SO₄ (3 times) andwater. To crystallize the product, the mixture was concentrated to 25 mland the mixture stirred. The product was obtained by filtering in theform of white crystals. Yield: 3.52 g (33%). ¹H NMR (300 MHz, DMSO) δ7.58 (dd, J=8.3, 0.7 Hz, 1H), 7.35 (dd, J=8.3, 2.4 Hz, 1H), 7.31 (d,J=2.2 Hz, 1H), 5.34 (s, 2H), 3.84 (s, 3H).

6-Hydroxyisobenzofuran-1(3H)-on

Nitrogen atmosphere, 6-methoxyisobenzofuran-1(3H)-on (3.00 g, 18.09mmol) was dissolved in anhydrous dichloromethane (100 ml). The resultingmixture was magnetically stirred and cooled in an ice bath for 10minutes. Then BBr3 (3.46 ml, 36.18 mmol) was added. The reaction mixturewas then heated to room temperature and stirred for 12 hours. Then 5 mlwater was added and the mixture was transferred to a separating funneland extracted with ethyl acetate (3×100 ml). The combined organicextracts were dried over magnesium sulphate, filtered and concentratedunder reduced pressure, wherein a white solid was obtained. Yield: 1.52g (56%). ¹H NMR (300 MHz, DMSO) δ 10.08 (s, 1H), 7.47 (dd, J=8.3, 0.6Hz, 1H), 7.19 (d, J=2.3 Hz, 1H), 7.16 (d, J=2.3 Hz, 1H), 7.10 (d, J=2.0Hz, 1H), 5.28 (s, 2H); ESI-MS: (calculated (MH)⁺: 151.04 g/mol, found:151.05 m/z).

5-azido-2-(hydroxymethyl)benzohydrazide

Dissolve 6-hydroxyisobenzofuran-1(3H)-on (0.43 g, 2.83 mmol) in 6 mldimethylformamide (DMF) and add hydrazine hydrate (1.42 ml, 28.26 mmol)and stir the solution at 100° C. for 3 hours. The DMF and hydrazinehydrate were removed under vacuum. The residue was purified by columnchromatography (CH₂Cl₂/MeOH, 9:1), wherein a light yellow powder wasobtained. Yield: 0.10 g (17%). Rf (CH₂Cl₂/MeOH, 9:1) 0.40; ¹H NMR (300MHz, DMSO) δ 10.08 (s, 1H), 9.73 (s, 1H), 7.47 (d, J=8.3 Hz, 1H), 7.18(dd, J=8.3, 2.3 Hz, 1H), 7.10 (d, J=2.3 Hz, 1H), 4.57 (d, J=5.7 Hz, 2H),4.50 (s, 2H), 3.33 (s, 2H), ESI-MS: (calculated (MH)⁺: 183.08 g/mol,found: 183.15 m/z).

Exemplary Purification with Capture Molecule (14)

The purification was performed with another peptide example 14(AKADEVSLHKWYG; SEQ ID NO: 10) and a linker of formula (14)(Fängermolekül 14) on commercially available, aldehyde-modified agarose(High Density Glyoxal, 6BCT from ABT).

Solid Phase Peptide Synthesis and Purification

The automated solid phase peptide synthesis was performed in 100 μmolbatches with a MultiPep RS peptide synthesis machine from Intavis AG.The synthesis was performed on a Wang-resin (1.0-1.4 mmol/g) from CarlRoth. Before the beginning of the synthesis, the corresponding amount ofpeptide synthesis resin was weighed in 5 mL syringe reactors (PE reactorfrom Intavis) and soaked in DMF. The weight of equivalents of amino acidwas refers to the initial loading of the resin used, unless otherwisestated.

Fmoc-Removal:

To remove the temporary Fmoc protecting groups, the resin was treatedonce for 5 min and once more for 8 min with 1500 μL piperidine/DMF (4:1)and subsequently washed seven times with 10.2 mL DMF and it wascontinued with the coupling of the Fmoc amino acid derivatives.

Coupling of Fmoc Amino Acid Derivatives:

A solution of 5 eq. amino acid in DMF (0.3 M) was pre-activated for 1min at room temperature with solutions of 4.5 eq. HCTU in DMF (0.3 M)and 10 eq. NMM in DMF (0.6 M) and then added to the resin. After 30 minreaction time, the resin was washed three times with 10.2 mL DMF and itwas continued with blocking the truncated sequences.

Blocking the Truncated Sequences:

The resin was treated twice for 5 min with 1.5 mL AC₂O/2,6-lutidine/DMFsolution (5:6:89) and subsequently washed seven times with 10.2 mL DMFeach.

Last Step Coupling of the Capture Molecule:

As the last step of the solid phase peptide synthesis, the capturemolecule (14) was coupled to the desired target peptide (50 μmol). Thesynthesis resin was mixed with a solution of 4 eq. capture molecule (0.3M), 6 eq. HOBt in DMF (0.4 M) and 4 eq. DIPEA in DMF (0.3 M) mixed.After a reaction time of 60 min, it was washed twice with 2 mL DMF each,twice with 2 mL CH₂Cl₂ and then washed again twice with 2 mL DMF.

Alternative Last Step of Acetylation of the Full-Length Peptide:

Analogously to the protocol for blocking the truncated sequences, thefull-length peptide as a control sample was also acetylated (peptideacetylated).

Release from Polymeric Support:

The resin was treated with 3 mL of a solution of 95% TFA, 2.5% water and2.5% triisopropylsilane. The synthesis resin was mixed with thiscleaving mixture and shaken for 3 h at room temperature. Afterwards, thecleaving solution was collected and the resin was washed twice with 1 mLTFA each. The cleaving solution was combined with the washing solutionsand concentrated by argon flow to approx. 1 mL volume. Afterwards, itwas precipitated with 10 mL cold diethyl ether and the precipitate wascentrifuged. The supernatant was discarded. In FIG. 4h (above) thechromatogram of the peptide without linker before purification is shownwith a purity of 45%.

Purification

The crude precipitate (about 5 μmol theoretical yield) was dissolved inconjugation buffer (0.1 M NH₄OAc, 0.1 M aniline, pH=3.8). If the mixturedid not dissolve completely, acetonitrile was added. In a 3 mL syringereactor with a 25 μm PE prefilter 400 μL (˜160 mg) agarose were added.The purification resin was then conditioned by washing 3 times withconjugation buffer (0.1 M NH₄OAc, 0.05 M aniline, pH=3.8). The peptidesolution was then added to the purification resin. Immobilization wasthen performed for 60 minutes. Afterwards, it was washed three timeswith conjugation buffer, three times with a 5 M urea solution, threetimes with 70% ethanol and finally five times with water. The mixturewas then treated basically with 5 v % NH₄OH in water to cleaveconjugated peptide from the resin. Lyophilization provides the peptideas a white flaky solid.

Proof of Immobilization

To provide clear evidence of immobilization, acetylated peptide andpeptide with bound linker were purified on modified as well as on pureagarose (6% B Agarose Bead STANDARD, ABT) with PEC in this experiment.The eluate after the left linker cleavage was assessed with UPLC-UV. Theresults showed that after cleavage, a significant signal of the productmass is only detected in peptide with bound linker on modifiedpurification resin. In addition, it can be seen that also with pureagarose about 2.3% of the product is obtained compared to modifiedagarose.

TABLE 3 Integrals of the product peak for the peptides afterpurification with modified and unmodified agarose Peptide linker Peptideacetylated Modified Modified Agarose agarose Agarose agarose Integral(μV*s) 17759 763044 197 131 Proportion 2.31% 100% 0.03% 0.02%

Regeneration of the Purification Resin

After peptide purification with the capture molecule (14), the originalaldehyde function of 1 remains blocked with the hydroxyl-modifiedcapture molecule. To make the purification resin available for a newpurification cycle, the resin must be regenerated and thus the aldehydefunction restored. This is achieved by shifting the equilibrium byadding aldehydes or ketones. Regeneration for repeated purificationcycles was demonstrated as follows:

Two purifications of peptide 14 were performed simultaneously(purification I). The purification resin was then washed four times eachwith a mixture of water, acetone and TFA (ketone, 49.95:49.95:0.1), orwater, acetaldehyde and TFA (aldehyde, 89.95:9.95:0.1) and five timeswith water for regeneration. Afterwards the purification including theconditioning was performed in the same way as described above. Theregeneration and purification was performed three times (purificationII-IV) and the lyophilized product was taken up in equal volumes ofwater, acetonitrile and TFA (69.9:29.9:1) and measured with UPLC-MS.

TABLE 4 Integrals of the product peak and percentage proportion for thepeptides after purification with modified agarose (purification I) andafter three regeneration cycles (purification II-IV). Ketone AldehydeIntegral Integral Purification (μV*s) Proportion Purity (μV*s)Proportion Purity I 4100144 100% 58.9% 2138955 100% 58.2% II 2044538 50% 58.5% 911365  43% 58.7% III 1719147  42% 57.8% 567140  27% 58.6% IV381717   9% 61.5% 576371  27% 59.8%

The results showed that the resin can be regenerated in both cases.Regeneration with a ketone only decreases significantly in the thirdcycle (purification IV). In contrast, in aldehyde regeneration, thepurification capacity remains at about a quarter of the initialcapacity, but decreases to this value already after the second cycle(purification III). In both experiments a purity of about 60% wasachieved, which remains constant during the regeneration cycles (examplechromatogram for purification I, aldehyde, FIG. 4h (below)).

We claim:
 1. Compound of the formulaX₁-L-X₂  (1), wherein X₁ is selected from

wherein each R¹ and R² is independently from each other selected from Hor B, wherein at least R¹ or R² is B, wherein R³ is selected from H orB, wherein B is an acid labile amine protecting group, wherein R⁴ isselected from H, C₁-C₁₂-alkyl or aryl, wherein the aldehyde or ketogroup may be protected by an acid labile protecting group, L is selectedfrom functional linkers, that are cleavable nucleophilically from X₂under basic conditions, in particular L is of the form -T-U—, wherein Tis a spacer between X₁ and U, wherein in particular T is selected fromsubstituted or unsubstituted —C₁-C₁₂-alkyl-, in particular C₁-C₆-alkyl,in particular C₁-C₃-alkyl, —R⁵—C(═O)—NH—R⁶—, —R⁵—C(═O)—O—R⁶—,—R⁵—C(═O)—O—, —C(═O)—O—R⁶—, —C(═O)—NH—R⁶—, —C(═O), —C(═O)—O—,—R⁵-phenyl-R⁶—, —R⁵-phenyl-, -phenyl-R⁶—, -phenyl-, wherein R⁵ and R⁶are independently from each other selected substituted or unsubstitutedC₁-C₁₂-alkyls, in particular C₁-C₆ alkyls, particularly C₁-C₃ alkyls,and wherein U is the cleavage activating part of the functional linker,wherein the activating part is formed to stabilize an anion formedduring an basic cleavage from X₂, X₂ is of the form —Y—Z, wherein Y isselected from —O—C(═O)— or —S(═O)₂—, and Z is an electron-withdrawingleaving group.
 2. Compound according to claim 1, wherein B is selectedfrom Boc (—C═OOtBu), trityl (—C(Ph)₃), Mmt (—C(Ph)₂C₆H₄OMe), DMT(—C(Ph)(C₆H₄OMe)₂), Cbz (—C═OOCH₂Ph), benzylideneamine (═CPh),phthalimides (═(CO)₂C₆H₄), p-toluenesulfonamides (—SO₂C₆H₄Me),benzylamine (—CH₂Ph), acetamides (—COMe), trifluoroacetamide (—COCF₃),Dde (1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-ethyl) and1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl (ivDde),wherein particularly B is Boc, and/or the acetal- or ketal protectinggroups are selected from

wherein r is 0 to 12, in particular 0, 1 or
 2. 3. Compound according toclaim 1, wherein T is selected from —C₁-C₁₂-alkyl-, —R⁵—C(═O)—NH—R⁶—,—R⁵—C(═O)—O—R⁶—, —R⁵—C(═O)—O—, —C(═O)—O—R⁶—, —C(═O)—NH—R⁶—, —C(═O)—,—C(═O)—O—, wherein R⁵ and R⁶ are independently from each other selectedC₁-C₆-alkyls, in particular C₁-C₂-alkyls, in particular T is selectedfrom —CH₂—, —CH₂—C(═O)—NH—(CH₂)₂—, —(CH₂)—C(═O)—O—(CH₂)₂—,—CH₂—C(═O)—O—, —C(═O)—O—, —C(═O)—O—(CH₂)₂—, —C(═O)—, in particular—CH₂—, —CH₂—C(═O)—NH—(CH₂)₂—, —C(═O)—O—(CH₂)₂—, —CH₂—C(═O)—O—, —C(═O)—.4. Compound according to claim 1, wherein U of the moiety T-U—Y isselected from the moieties according to the formulas (5), (6), (7), (8),(9), (10) and (11), in particular from (5), (6), (8), (9) and (10),

wherein R⁸ is selected from C₁-C₆-alkyl, CF₃, CH₂CF₃,

in particular from Boc-Lys(Boc)-, wherein R⁷ _(n), R⁹ _(m), R¹⁰ _(p),R¹¹ _(q), R¹³ _(r) and R¹⁴ _(s) is selected from C₁-C₆-alkyl or —Iand/or -M-effects generating substituents, in particular C₁-C₃-alkyls,—F, —Cl, —Br, —I, —CN —NO₂, —N₃, —CF₃, —SO₃H, —CO₂H, wherein n equals 0,1, 2, 3 or 4, in particular n is 0 oder 1, in particular 0, wherein mequals 0, 1, 2 or 3, in particular m is 0 oder 1, in particular 0,wherein p equals 0, 1, 2, 3 or 4, in particular p is 0 oder 1, inparticular 0, wherein q equals 0, 1, 2 or 3, in particular q is 0 oder1, in particular 0, wherein r equals 0, 1, 2 or 3, in particular r is 0oder 1, in particular 0, wherein s equals 0, 1, 2 or 3, in particular sis 0 oder 1, in particular
 0. 5. Compound according to claim 1, whereinZ is selected from the group —F, —Cl, —Br, —I, —N₃, —SR¹², —OCF₃,—OCH₂CF₃, —OSO₂CF₃, —SO₂C₆H₄CH₃, —SO₂CF₃, —SO₂CH₃

in particular —Cl,

in particular

wherein R¹² is a C₁-C₆-alkyl-, an aryl- or a benzyl residue.
 6. Compoundaccording to claim 1, wherein X₁ is a moiety of formula (2) or (3), inparticular of formula (3), wherein R³ is H, R¹ and R² comprise a Bocprotecting group or R¹ is H and R² is a Boc protecting group, wherein inparticular R¹ is H and R² is a Boc protecting group.
 7. Compoundaccording to claim 1, wherein Y is of the form —O—C(═O)—.
 8. Compoundaccording to claim 1, wherein T is of the form —(CH₂)—C(═O)—NH—(CH₂)₂—,—(CH₂)—C(═O)—O—(CH₂)₂—, —C(═O)—O—(CH₂)₂—, in particular —C(═O)—O—(CH₂)₂—and —(CH₂)—C(═O)—NH—(CH₂)₂—, in particular —(CH₂)—C(═O)—NH—(CH₂)₂—,wherein U is a moiety of formula (5) or (6), in particular of formula(6),


9. Compound according to claim 1, wherein T is of the form—CH₂—C(═O)—O—, —C(═O)—O—, in particular —CH₂—C(═O)—O—, wherein U is amoiety of formula (7),

wherein R⁷ is selected from C₁-C₆-alkyl or —I and/or -M-effectgenerating substituents, in particular C₁-C₃-alkyl, —F, —Cl, —Br, —I,—CN —NO₂, —N₃, —CF₃, —SO₃H, —CO₂H wherein n equals 0, 1, 2, 3 or 4, inparticular 0 or 1, in particular
 0. 10. Compound according to claim 1,wherein T is of the form —CH₂—, wherein U is a moiety of formula (8),

wherein R⁸ is Boc-Lys(Boc) and r equals
 0. 11. Compound according toclaim 1, wherein T is of the form —CH₂—, —(C═O)—, in particular —CH₂—,wherein U is a moiety of formula (9), wherein s equals 0,


12. Compound according to claim 1, wherein T is of the form —C(═O)—,wherein U is a moiety of formula (10), wherein m equals 0,

wherein Y is of the form —SO₂—, and wherein Z is Cl.
 13. Use of acompound according to claim 1 as a connection between the N-terminalamino group of a full-length peptide and a solid phase.
 14. A compoundof formula X₁-L-Y-PEP (12), wherein X₁, L and Y is defined according toclaim 1, and wherein PEP comprises a full-length peptide bound to X₂′via its N-terminus.
 15. A compound of formula D-X₁′-L-Y-PEP (13),wherein D is a surface-modified solid support, which is characterized inthat the surface is modified by synthetic or natural polymers, inparticular modified polysaccharides, in particular aldehyde- orhydrazine-modified sepharose/agarose or cellulose, wherein X₁′ is of theform —NH—O—, —NH—NH— or —C(═O)—, wherein L, Y and PEP are definedaccording to claim
 12. 16. Method for the purification of peptides, inparticular of peptides prepared by solid phase peptide synthesis (SPPS),comprising the following steps: i. contacting a composition of afull-length peptide to be purified and at least one impurity, inparticular at least one acetylated truncated sequence, with a capturecompound according to claim 1, and subsequent reaction to a compound offormula (12), ii. cleavage of the acid labile protecting groups byaddition of an acid, iii. contacting the composition of ii. with asurface-modified solid support, wherein a covalent hydrazone or oximebond is formed between the capture compound and the solid support, and acompound of formula (13) is provided, iiia. optionally, cyclization ofthe moiety PEP of the compound of formula (13) by oxidation of two ormore residues bearing a nucleophilic thiol towards a disulfide bridgeusing an oxidizing agent, iv. cleavage of the optionally modifiedfull-length peptide from the solid support.
 17. Method according toclaim 16, wherein the solid support comprises on its surface thefunctional groups aldehyde, in particular —O—CH₂—CHO, ketone,hydroxylamine, in particular —ONH₂, and hydrazine, in particular —N₂H₃.18. Method according to claim 16, wherein after or during cleavage ofthe full-length peptide from the solid support, the solid support D iscleaved from the residue X₁-L of the capture compound and the solidsupport is regenerated.
 19. The method according to claim 16, whereinthe cyclization is performed in the presence of air, particularly in thepresence of oxygen, and/or by a basic aqueous solution with a pH >7 topH 8.5, in the presence of an oxidative additive, in particular anoxidative additive selected from DMSO, iodine, N-chlorosuccinimide,—Tl(OAc)₃, —Tl(CF₃COO)₃, —CH₃SiCI₃-Ph(SO)Ph,[Pt(ethylenediamine)₂Cl₂]Cl₂, 2,2′-Dithiobis(5-nitropyridine),5,5′-dithiobis-(2-nitrobenzoic acid), trans-[Pt—(CN)₄Cl₂]²⁻,glutathione-glutathione disulfide, K₃Fe(CN)₆.
 20. The method accordingto claim 16, wherein the number of nucleophilic thiols in the peptide iseven, particularly the peptide comprises 2 to 10 amino acids comprisinga nucleophilic thiol, and/or the peptide comprises at least two aminoacids independently selected from cysteine, homocysteine orpenicillamine.